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Publication numberCN87102927 A
Publication typeApplication
Application numberCN 87102927
Publication date27 Apr 1988
Filing date18 Mar 1987
Priority date19 Mar 1986
Also published asDE3789679D1, DE3789679T2, EP0238329A2, EP0238329A3, EP0238329B1, US5175082
Publication number87102927.8, CN 87102927, CN 87102927 A, CN 87102927A, CN-A-87102927, CN87102927, CN87102927 A, CN87102927.8, CN87102927A
Inventors亚历克·约翰·杰弗里斯
Applicant帝国化学工业公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Improvements in genetic probes
CN 87102927 A
Abstract  translated from Chinese
可使用其中每一个都是对信息性基因位点特异的多核苷酸探针来定性基因组DNA的试验样品。 Polynucleotide probes can be used which are each gene locus information specific to the qualitative test sample of genomic DNA. 可使用能根据一个以上多态性小随体区或高可变位点鉴定DNA的探针制备这样的探针。 The probe can be used for preparing one or more polymorphic minisatellite region or hypervariable locus identification of such DNA probes. 本发明的多核苷酸和探针可用于遗传定性、父亲或母亲身份试验,特别是可用于法医学中。 Polynucleotides and probes of the invention can be used for genetic characterization, the identity of the father or the mother tests are particularly useful in forensic medicine.
Claims(40)  translated from Chinese
1.制备能够杂交到含有一小随体之DNA片段上的多核苷酸的方法,所说的小随体是对基因组中的特定区域或位点特异的,上述区域或位点具有至少为3(100)的等位基因变异(如说明书所定义的)。 1. Preparation of a polynucleotide capable of hybridizing to a method comprising the DNA fragment of a minisatellite, said minisatellite genome is a specific area or site-specific, said region or locus having at least 3 (100) allelic variation (as defined in the specification). 该方法包括克隆一个通过将基因组DNA的片段与一多核苷酸探针杂交而鉴定的DNA片段,该多核苷酸探针能够参考一个以上的多态性小随体区域或高可变位点以鉴别DNA;由之制备能够杂交到一含小随体之DNA片段上的多核苷酸,该小随体对于基因组中的某特定区域或位点是特异的,而所说的区域或位点具有至少为3(100)的等位基因变异(如说明书中所定义的)。 The method comprises cloning a fragment of genomic DNA by the hybridization with a polynucleotide probe and a DNA fragment identified, the polynucleotide probe can refer to more than one polymorphic minisatellite region or hypervariable locus to identify DNA; from the preparation capable of hybridizing to a DNA fragment containing the polynucleotide of the minisatellite of the minisatellite genome for a particular region or locus is specific, and said region or locus having at least 3 (100) is an allelic variation (as herein defined).
2.根据权利要求1的方法,其中所说的DNA片段,其含有一源自小随体区域高可变位点的小随体,所说的区域或位点具有至少为3的等位基因变异(如说明书中所定义的)。 2. A method according to claim 1, wherein said DNA fragment containing a minisatellite region derived from hypervariable minisatellite locus, said region or locus having at least 3 of the allele variation (as herein defined).
3.制备能杂交到含有一小随体之DNA片段上的多核苷酸的方法,所说的小随体是对基因组中的特定区域或位点特异的,上述区域或位点具有至少为3的多态性程度(如说明书中所定义的)。 3. Preparation method capable of hybridizing to a DNA fragment containing the polynucleotide of minisatellite on, said minisatellite genome is a specific area or site-specific, said region or locus having at least 3 the degree of polymorphism (e.g., as defined in the specification). 该方法包括克隆一个通过将基因组DNA的片段与一多核苷酸探针杂交而鉴定出的DNA片段,该多核苷酸探针能够参考一个以上的多态性小随体区域高可变位点以鉴别DNA;由之制备能够杂交到一含小随体之DNA片段上的多核苷酸,该小随体对于基因组中的某特定区域或位点是特异的,所说的区域或位点具有至少为3的多态性程度(如说明书中所定义的)。 The method comprises cloning a fragment of genomic DNA by the hybridization with a polynucleotide probe and identified DNA fragment, the polynucleotide probe can refer to more than one polymorphic minisatellite region hypervariable locus to identify DNA; from the preparation capable of hybridizing to a DNA fragment containing the polynucleotide of the minisatellite of the minisatellite genome for a particular region or locus is specific, said region or locus having the degree of polymorphism of at least 3 (as hereinbefore defined).
4.根据上述任一权利要求的方法,其中多核苷酸探针包含同时包括有己标记或标志组件的、至少含3个串联重复(在所有串联重复之序列间平均至少有70%的同源性)序列的多核苷酸,这些序列与基因组的小随体区是同源的,其相似程度为能使探针杂交到用限制性核苷酸内切酶切断样品DNA所得到的一相应之DNA片段上,其中:a)每次重复都含有一个核心,该核心与来自不同之基因位点的多个小随体中存在之有相似长度的相符核心区至少有70%的同源性(即70%相符);b)该核心长度为6至16个核苷酸;c)对该核心没有贡献的重复单元中核苷酸的总数不超过15个。 4. A method according to any preceding claim, wherein the polynucleotide probe comprises simultaneously comprises mark or flag has component, containing at least three tandem repeats (between the tandem repeat sequence of all average of at least 70% homology ) polynucleotide sequences, these sequences minisatellite region that is homologous with the genome, and its degree of similarity to enable probe hybridizes to a nucleotide with a restriction enzyme cutting of DNA samples of a respective DNA obtained fragment, wherein: a) each repeat contains a core, the core with a plurality of small differences from the minisatellite loci in the presence of similar length consistent core area at least 70% homology (i.e. 70% match); b) the core length from 6 to 16 nucleotides; Total c) does not contribute to the core repeat units of no more than 15 nucleotides.
5.根据上述权利要求之任一项的方法,其中多核苷酸探针包含序列(包括其互补序列)(A)AGGGCTGGAGG(其中T是T或U,(A)可有可无),或序列(包括其互补序列)AGAGGTGGGCAGGTGG(其中T是T或U)的至少三次串联重复;所有串联重复的序列之间至少有70%相符合。 According to any of the preceding claims, wherein the probe comprises the polynucleotide sequence (including complementary sequences) (A) AGGGCTGGAGG (wherein T is T or U, (A) optional), or the sequence (including complementary sequences) AGAGGTGGGCAGGTGG (wherein T is T or U) of at least three tandem repeat; at least 70% conform to the sequence between all tandemly repeated.
6.根据权利要求5的方法,其中多核苷酸探针包含有选自:AGGAATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TGTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9(其中Y是C、T或U,X是G或C,R是A或G,T是T或U)的任何一个序列或其串联重复,或与之互补的序列。 6. The method according to claim 5, wherein the polynucleotide probe selected from the group comprising: AGGAATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TGTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9 (wherein Y is C, T or U, X is G or C, R is A or G, T is T or U) or a tandem repeat of either sequence, or a sequence complementary thereto.
7.根据权利要求1-5中任一项的方法,其中所说的片段含有一得自小随体区或高可变位点的小随体,该区域或位点可用含有序列式3之序列或其串联重复、或其互补序列的多核苷酸探针检测出来,该方法是在能有效地鉴定单一位点的杂交条件下实施的。 7. A method according to any one of claims 1-5, wherein said fragment contains Gains childhood minisatellite region or hypervariable minisatellite locus which region or locus is available containing the sequence of formula 3 sequence or a tandem repeat, or a complementary polynucleotide probe sequences detected, the process is effective to identify a single site of hybridization conditions under implementation.
8.根据权利要求1-5中任一项的方法,其中所说的片段含有一来自小随体区或高可变位点的小随体,所说的区域或位点可用包含选自序列式4至9的任一序列或其串联重复,或与之互助之序列的多核苷酸探针检测出来,该方法是于能有效地鉴定某单一位点的杂交条件下实施的。 8. A method according to any one of claims 1-5, wherein said fragment contains a minisatellite from a minisatellite region or hypervariable locus, said region or locus comprising a sequence selected from the available of formula 4-9 according to any one sequence or tandem repeats, or with a polynucleotide probe sequence mutual assistance detected, the process is to identify hybridization conditions effective to a point under a single embodiment.
9.根据前述权利要求中之任一项的方法,其中多核苷酸序列是与含小随体之DNA片段中的小随体同源的,且相符程度为能使该核苷酸序列与之杂交。 According to any one of the preceding claims, wherein the polynucleotide sequence is a DNA fragment containing the body of the minisatellite in the minisatellite homologous, and enables the degree of coincidence is a nucleotide sequence with hybridization.
10.一被分离或克隆形式的多核苷酸,它包含有对单一小随体区或高可变位点特异的並与之同源的核苷酸序列,其相符程度为能使该核苷酸序列杂交到用限制性核苷酸内切酶切断样品基因组DNA所得到的相应DNA序列上,所说的DNA片段包含一来自所说的小随体区或高可变位点的小随体,其中:1)所说的区域或位点具有至少为3(100)的等位基因变异(根据说明书中的定义);2)所说的区域或位点可用包含有序列(包括其互补序列):(A)AGGGCTGGAGG 1(其中T是T或U,(A)可有可无)或序列(包括其互补序列):AGAGGTGGGCAGGTGG 2(其中T是T或U)之至少三次串联重复的多核苷酸探针检测出来。 10. A be isolated or cloned forms of the polynucleotide, which comprises a nucleotide sequence of a single minisatellite region or hypervariable locus-specific and homologous thereto, which enables the degree of coincidence of nucleoside acid sequence that hybridizes to a nucleotide with a restriction enzyme cutting the corresponding DNA sequences of genomic DNA samples obtained, said DNA fragment containing a minisatellite from said region or hypervariable locus minisatellite wherein: 1) said region or locus having at least 3 (100) of the allelic variation (as defined herein); and 2) said region or locus is available containing sequences (including their complementary sequences ) :( A) AGGGCTGGAGG 1 (wherein T is T or U, (A) optional) or the sequence (including complementary sequences): AGAGGTGGGCAGGTGG 2 (wherein T is T or U) of at least three tandem repeats of the polynucleotide acid probes detected.
11.根据权利要求10的多核苷酸,其中DNA片段含有来自小随体区域或高可变位点的一小随体,所说的区域或位点具有至少为3(500)的等位基因变异(根据说明书中所述的定义)。 11. The polynucleotide of claim 10, wherein the DNA fragment contains a minisatellite from a minisatellite region or hypervariable locus, said region or locus having at least 3 (500) allele variation (according to the definition described in the specification).
12.一被分离或克隆形式的多核苷酸,它包含有对单一小随体区或高可变位点特异的並且与之同源的核苷酸序列,其相符程度足能使该核苷酸序列杂交到用某限制性核酸内切酶切断样品基因组DNA所得到的相应DNA片段上,所说的DNA片段含有一来自所说之小随体区或高可变位点的小随体,其中:1)所说的区域或位点具有至少为3的多态性程度(如说明书中所定义的);2)所说的区域或位点可用含有序列(包括其互补序列):(A)AGGGCTGGAGG 1(其中T是T或U,(A)可有可无)或序列(包括其互补序列):AGAGGTGGGCAGGTGG 2(其中T是T或U)的至少三次串联重复的多核苷酸探针检测出来。 12. A be isolated or cloned forms of the polynucleotide, which contains a single minisatellite region or hypervariable locus-specific and the nucleotide sequence homologous thereto, which enables the degree of coincidence sufficient nucleoside acid sequence to hybridize with a certain restriction endonuclease cut on the corresponding DNA fragment of genomic DNA samples were obtained, said DNA fragment containing a minisatellite from a minisatellite region or hypervariable locus of the said, wherein: 1) said region or locus having a degree of polymorphism of at least 3 (as defined herein); and 2) said region or locus is available containing sequences (including their complementary sequences) :( A ) AGGGCTGGAGG 1 (wherein T is T or U, (A) optional) or the sequence (including complementary sequences): AGAGGTGGGCAGGTGG 2 (wherein T is T or U) at least three tandem repeats of a polynucleotide probe assay come out.
13.根据权利要求10-12中任一项的多核苷酸,其中DNA片段含有来自一小随体区或高可变位点的小随体,所说的区域或位点可特异地被含有选自:AGGATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TGTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9(其中Y是C、T或U,X是G或C,R是A或G,T是T或U)的任何一个序列或其串联重复或其互补序列的多核苷酸探针鉴定出来。 10-12 according to any one of the polynucleotide of claim, wherein the DNA fragment contains a minisatellite from a minisatellite region or hypervariable locus, said region or locus may specifically be contained is selected from: AGGATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TGTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9 (wherein Y is C, T or U, X is G or C, R is A or G, T is T or U), or any of a tandem repeat or its complementary sequence polynucleotide probe sequences identified.
14.根据权利要求10-12中任一项的多核苷酸,其中所说的DNA片段含有一来自小随体区或高可变位点的小随体,所说的区域或位点可由含有序列式3之序列或其串联重复序列的多核苷酸探针检测出来。 10-12 according to claim any one of the polynucleotide, wherein said DNA fragment containing a minisatellite from a minisatellite region or hypervariable locus, said region or locus may contain 3 of the sequence of formula or a tandem repeat sequence of a polynucleotide probe detected.
15.根据权利要求10-12之任一项的多核苷酸,其中所说的DNA片段含有一来自小随体区或高可变位点的小随体,所说的区域或位点可用含有选自序列式4至9中任一序列之串联重复序列的多核苷酸探针检测出来。 According to any one of claims 10-12 of a polynucleotide, wherein said DNA fragment containing a minisatellite from a minisatellite region or hypervariable locus, said region or locus containing an a sequence selected from any one of formulas 4-9 tandem repeats of a sequence of the polynucleotide probe detected.
16.根据权利要求10-15中任一项的多核苷酸,其中核苷酸序列是与DNA片段的小随体同源的,其相符程度为能使该核苷酸序列与之杂交。 10-15 according to claim any one of the polynucleotide, wherein the nucleotide sequence is minisatellite DNA fragments homologous, which enables the degree of coincidence of the nucleotide sequence hybridize.
17.根据权利要求14的多核苷酸,其中核苷酸序列包含序列式3的序列或其串联重复(所有呈串联重复的序列之间平均至少有70%是一致的)。 17. The polynucleotide of claim 14, wherein the nucleotide sequence comprises the sequence of formula 3 or a tandem repeat (between all tandemly repeated sequences was an average of at least 70% is the same).
18.根据权利要求15的多核苷酸,其中核苷酸序列包含选自序列式4至9的任一序列或其串联重复(所有呈串联重复的序列之间平均至少有70%是一致的)或与之互补的序列。 18. The polynucleotide of claim 15, wherein the nucleotide sequence comprises a sequence selected from any of formula 4-9, or a tandem repeat of a sequence (between all tandemly repeated sequences had an average of at least 70% identical) or a sequence complementary thereto.
19.根据权利要求17或18的多核苷酸,其中所有呈串联重复的序列之间平均至少有80%是一致的。 19. The polynucleotide of claim 17 or 18, wherein between all tandemly repeated sequences was an average of at least 80% identical.
20.根据权利要求19的多核苷酸,其中所有呈串联重复的序列之间平均至少有90%是一致的。 20. The polynucleotide of claim 19, wherein between all tandemly repeated sequences was an average of at least 90% identical.
21.根据权利要求20的多核苷酸,其中串联重复的序列是准确重复的。 21. The polynucleotide of claim 20, wherein the tandem repeat sequence is exact duplicate.
22.制备多核苷酸的工艺,其首先是按照权利要求1-9之任一项中所限定的方法制备多核苷酸,或者说制备权利要求10-21之任一项中所限定的多核苷酸,该工艺包括用微生物学手段复制己克隆的材料。 22. A process for preparing a polynucleotide, which is the first of any one of 1-9 in accordance with the method as defined in claim preparation polynucleotide of any one of claims 10-21 polynucleotide as defined in claim or prepared acid, the process comprising replication with microbiological methods have cloned material.
23.制备多核苷酸的工艺,其首先是按照权利要求1-9之任一项中所限定的方法制备多核苷酸,或者是经直接合成制备权利要求10-21之任一项中所限定的多核苷酸。 23. A process for preparing a polynucleotide, which is the first of any one of 1-9 in accordance with the method as defined in claim polynucleotide preparation, or prepared by direct synthesis of any one of claims 10-21 as defined polynucleotide.
24.多核苷酸探针,其包含权利要求10-21之任一项中所限定的多核苷酸,或者按照权利要求1-9之任一项中所限定的方法制备的多核苷酸,所说的多核苷酸具有一标记的或标志组件。 24. A polynucleotide probe which comprises any one of claims 10-21 as defined in the polynucleotide or the polynucleotide according to any one of claims 1-9 prepared by the method as defined in, and the said polynucleotide having a labeled or marker component.
25.根据权利要求24的多核苷酸,其中多核苷酸整个是单股形式的。 25. The polynucleotide of claim 24, wherein the polynucleotide is single-stranded form of the whole.
26.制备权利要求24或25中所限定之多核苷酸的方法,其包括标记或标志如权利要求10-21之任一项中所述的、或按照权利要求1-9之任一项中所限定的方法制备的多核苷酸。 26. 24 or 25 in the preparation of the polynucleotide of the method defined in claim, which comprises a mark or logo as in any one of claims 10-21, wherein, according to any one of claims 1-9 or of one of prepared by the method as defined polynucleotide.
27.制备能够杂交到一DNA片段上之多核苷酸的方法,该DNA片段含有一对基因组中特殊区域或位点特异的小随体,所说的区域或位点具有至少为3(100)的等位基因变异(如说明书中所定义的)。 27. Preparation of DNA capable of hybridizing to a polynucleotide fragment of a method, the DNA fragment containing the gene pair in a special area or site-specific minisatellite, said region or locus having at least 3 (100) allelic variation (as herein defined). 该方法包括克隆一通过以基因组片段与某多核苷酸探针杂交而鉴定的DNA片段,所说的多核苷酸探针是如权利要求24或25中所要求的,或是按权利要求26的方法制备的,进而由之制备一能够杂交到含小随体之DNA片段上的多核苷酸,该小随体是对基因组上某特殊区域或位点特异的,所说的区域或位点具有至少为3(100)的基因基因变异(如说明书中所定义的);该方法是于能使探针参考一个以上多态性小随体区或高可变位点以鉴别DNA的杂交条件下实施的。 The method comprises cloning a genomic fragment by a polynucleotide probe hybridization to identify DNA fragments of said polynucleotide probes are 24 or 25 as claimed in claim, or according to claim 26 prepared by the method, and further by the preparation of a polynucleotide capable of hybridizing to the DNA fragment containing the minisatellite of on, the minisatellite is specific for a specific region or locus on the genome, said region or locus having at least 3 (100) of the gene mutation (such as defined in the specification); the method is to make a reference probe above polymorphic minisatellite region or hypervariable locus to identify DNA hybridization conditions implementation.
28.一被分离或克隆形式的多核苷酸,其包含一对单一小随体区或高可变位点特异的、並且与之同源的核苷酸序列,其同源相符程度为能使该核苷酸序列杂交到用限制性核酸内切酶切断样品基因组DNA所得到的相应之DNA片段上,所说的DNA片段含有一得自所说之小随体区或高可变位点的小随体,其中:1)所说的区域或位点具有至少为3(100)的等位基因变异(如说明书中所定义的),並且2)所说的区域或位点是可用权利要求24或25中所述的多核苷酸探针或者按权利要求26之方法制备的多核苷酸探针检测的。 28. A clone is isolated or in the form of a polynucleotide, comprising a single minisatellite region or hypervariable locus-specific, and the nucleotide sequence homologous thereto, which enables the degree of homology is consistent The nucleotide sequence that hybridizes to a nucleic acid with a restriction enzyme cutting of the DNA fragment corresponding genomic DNA samples were obtained, said DNA fragment containing the gains and from said minisatellite region or hypervariable locus minisatellite, wherein: 1) said region or locus having at least 3 (100) of the allelic variation (as herein defined), and 2) said region or locus is available to claim 24 or 25 polynucleotide probe or in the polynucleotide probe according to claim 26. The method for preparing detected.
29.对权利要求27中限定之方法,或权利要求28中所述的多核苷酸的修变,其中所说的区域或位点具有至少为3的多态性之程度。 Polynucleotides modifier of the method described in 28 to 27 defined in the claim 29., or the claims, wherein said region or locus having a degree of polymorphism of at least 3 is.
30.参考一个或多个对照物定性基因组DNA之试验样品的方法,该方法包括使用一种或多种不至于切断相应于串联重复之序列中任何相关限度的限制酶切断样品DNA;用多核苷酸或多核苷酸探针探查该DNA片段-所说的多核苷酸或多核苷酸探针含有对单一小随体区或高可变位点特异的並与之同源的核苷酸序列,其序列相符程度应能使该核苷酸序列杂交到含得自所说单一小随体区或高可变位点之小随体的相应DNA片段上;检测已杂交的DNA之片段,並将已杂交的片段与所说的一个或多个对照物进行比较,其中:1)所说的小随体区或高可变位点具有至少为3(100)的等位基因变异,並且2)所说的区域位点是可以用能够参考一个以上多态性小随体区或高可变位点鉴定DNA的多核苷酸探针探测到的。 30. A reference to one or more of the qualitative controls genomic DNA of the test sample, the method comprising the use of one or more not cut corresponding to the tandem repeat sequence of any relevant limit restriction enzyme cut the sample DNA; with polynucleotide acid or polynucleotide probes of the probe DNA fragment - of said polynucleotide or polynucleotide probe comprising a nucleotide sequence of a single minisatellite region or hypervariable locus-specific and homologous thereto, Its sequence should enable the extent to which nucleotide sequence that hybridizes to a solution obtained from said small single minisatellite region or hypervariable locus of the DNA fragments with the corresponding body; detect the hybridized DNA fragments, and hybridized with a fragment of said one or more controls are compared, wherein: 1) said minisatellite region or hypervariable locus has at least 3 (100) of allelic variation, and 2) said region sites can be used with reference to a polynucleotide probe capable of more than one polymorphic minisatellite region or hypervariable locus detected by the identification of the DNA.
31.参考一个或多个对照物以定性基因组DNA之试验样品的方法,该方法包括用一种或多种不至于切断相应于串联重复之序列到任何相关程度的限制酶切断样品DNA;用一多核苷酸或多核苷酸探针探查该DNA片段-所说的多核苷酸或多核苷酸探针含有对单一小随体区或高可变位点特异的並与之同源的核苷酸序列,其序列相符程度应能使该核苷酸序列杂交到含有得自所说单一小随体区或高可变位点之小随体的相应DNA片段上;检测已杂交的DNA之片段並将已杂交的片段与所说的一个或多个对照物进行比较,其中:1)所说的小随体区或高可变位点具有至少为3个的多态性程度;並且2)所说的区域或位点是可以用能够参照一个以上多态性小随体区或高可变位点鉴定DNA的多核苷酸探针探测到的。 31. A reference to one or more controls to the method of the test sample of genomic DNA of the qualitative, which method comprises one or more not cut corresponding to the tandem repeat sequence to the extent of any relevant restriction enzyme cut the sample DNA; with a a polynucleotide or polynucleotide probe of the probe DNA fragment - of said polynucleotide or polynucleotide probe comprising a single minisatellite region or hypervariable locus-specific homologous nucleosides and with detection of the hybridized DNA fragments; acid sequence, which sequence enables the extent to which should hybridize to the nucleotide sequence containing DNA from the corresponding fragment of said small single minisatellite region or hypervariable locus and with the body and hybridized with a fragment of said one or more controls are compared, wherein: 1) said minisatellite region or hypervariable locus has at least three degrees of polymorphisms; and 2) said region or locus are able to be used with reference to one or more polynucleotide probes polymorphic minisatellite region or hypervariable locus detected by the identification of the DNA.
32.根据权利要求30或31的方法,其中所用的多核苷酸或多核苷酸探针是由权利要求10-21中之任一项所限定的或者是依照权利要求1-9之任一项的方法制备的。 32. The method according to claim 30 or claim 31, wherein the polynucleotide or polynucleotide probe used is any one of 10 to 21 as defined by the claims, or in accordance with any one of claims 1-9 of a The method of preparation.
33.根据权利要求30-32之任一项的方法,其中样品DNA是人DNA。 33. The method according to any one of 30-32 of claim wherein the sample DNA is human DNA.
34.根据权利要求30-33之确证试验样品供体与一个或多个对照样品供体为完全一致或不一致的方法,其中对照样品是作了相似地酶切的,並将得自各个样品的杂交片段进行比较。 34. Claim 30-33 of confirmatory test sample donor with one or more control sample donors fully concordant or discordant, wherein the control sample is made similarly digested, and each sample was obtained from hybridizing fragment were compared.
35.根据权利要求30-33的确证试验样品供体与一个或多个对照样品供体之间家族关联的方法,其中对照样品是经过了相似地酶切的,並将得自各个样品的杂交片段进行比较。 35. The claim 30-33 confirmatory test sample donor with one or more methods familial association between the control sample donors, in which the control sample is similarly been digested, and the respective samples obtained from hybridization fragments were compared.
36.根据权利要求30-35之任一项的方法,其中探查是使用至少两个如权利要求24或25中所述的多核苷酸探针完成的,使用所说的探针进行一系列试验或单一试验。 36. A method according to any of claims 30-35, wherein the probe is used, the use of at least two of said probe polynucleotide probe as described in 24 or claim 25 performed a series of experiments or a single test.
37.根据权利要求30或31的诊断遗传性疾病、异常或特性的方法,其包括用至少一个如权利要求22或33中所述的多核苷酸探针探查试验样品,所说的探针-其並不一定携带被标记或标志组件-是与特殊的遗传性疾病、异常或特性相联系的。 37. The diagnosis 30 or 31 of hereditary disease, disorder, or characteristic of a method claim, comprising using at least one polynucleotide probe as claimed in claim exploration test sample 22 or 33, wherein said probe - it does not necessarily carry the labeled or marker component - is the specific genetic disease, disorder or characteristic associated.
38.基本上如本申请之说明书所述的並根据任一个实施例的多核苷酸探针。 38. A substantially as herein described and claimed in the present application of the polynucleotide probe according to any one of the embodiments.
39.基本上如本申请之说明书所述的並根据pλg3、λMS1、λMS31、λMS32、λMS8(1)、λMS32、λMS8(1)、λMS8(2)和λMS43中之一的多核苷酸探针。 39. The substantially as described in the specification of the present application and according to pλg3, λMS1, λMS31, λMS32, λMS8 (1), λMS32, λMS8 (1), λMS8 (2) and polynucleotide probe λMS43 in one.
40.基本上如本申请说明书所述的遗传定性的方法。 40. The substantially as described in the specification of this application genetic qualitative methods.
Description  translated from Chinese

本发明总地涉及多核苷酸和DNA及RNA探针、它们的制备以及它们在遗传特性鉴定中的应用。 The present invention generally relates to polynucleotides and DNA and RNA probes, their preparation and their use in the identification of genetic characteristics. 这些用途例如可以包括证实人、动物或植物的来源,因而本发明的多核苷酸和探针可以在例如父亲身份争异或法医学中或者在遗传病或遗传素因的予防、诊断和治疗方面得到应用。 These uses may include, for example, confirm that the source of human, animal or plant, and thus the polynucleotides and probes of the invention may be, for example a preventive, diagnostic and therapeutic aspects of contention paternity or forensic medicine or in heterologous genetic disease or genetic predisposition has been applied .

在英国专利申请NO8525252(公开号2166445)中描述了多种DNA序列,这些序列可以用来作为探针在人和动物基因组内的许多多态性位点处分别杂交,使得能够产生由标记的不同分子量的带构成的“指纹图”。 Described in British Patent Application NO8525252 (Publication No. 2166445) in a variety of DNA sequences, these sequences can be used as a probe at a number of polymorphic sites within the human and animal genome hybridization respectively, enables the production of different markers by molecular weight band constitutes the "fingerprint." 这种指纹图总体上是有关的个体的特征,通过个体的谱系可以追溯这些不同谱带的来源,在某些情况下其来源可以假定为与某些遗传病有联系。 This fingerprint is generally related to the characteristics of the individual, the individual's lineage can be traced by these bands of different sources, in some cases the source can be assumed in connection with certain genetic diseases.

本发明是基于这样一个发现,即通过使用多位点探针在基因组中可以识别信息性基因位点,并且可以制备出一些多核苷酸及多核苷酸探针,其中每个对于这样的单一的信息性基因位点是特异的。 The present invention is based on the discovery that by using the multi-locus probes can be identified in the genome informative genetic locus, and can be prepared a number of polynucleotides and polynucleotide probes, wherein each for such single informative loci is specific.

迄今为止,在人体DNA中只发现了数量有限的高可变位点,这些位点包括:胰岛素基因的5′小随体(minisatellites)(Bell、Selby和Rutter,1982);C-Ha-rasl基因的3′小随体(Capon等,1983),Ⅱ型胶原基因(Stoker等,1985)和ξ2与ψξ1珠蛋白基因之间的区域(Goodbourn等,1983),以及由染色体14之长臂的终端区得到的无名DNA克隆所限定的D14S1位点(Wyman和White,1980;Balazs等,1982;Wyman、Wolfe和Botstein,1984)。 So far, only found in human DNA in a limited number of hypervariable loci, these sites include: 5 'insulin gene minisatellite (minisatellites) (Bell, Selby and Rutter, 1982); C-Ha-rasl 3 'gene minisatellite (Capon et al., 1983), type Ⅱ collagen gene (Stoker et al., 1985) and ξ2 between ψξ1 globin gene region (Goodbourn et al., 1983), as well as by the long arm of chromosome 14 of the anonymous DNA clone obtained terminal area defined D14S1 locus (Wyman and White, 1980; Balazs, etc., 1982; Wyman, Wolfe and Botstein, 1984). 这些小随体的变异性有实质性差异,其范围从在胶原高可变区检测到的仅仅6个不同的等位基因(Sykes、Ogilvie和Wordsworth,1985)到在D14S1位点处的80多个(Balazs等,1986)。 Variability of these minisatellites differ materially, ranging from the collagen hypervariable regions detected only six different alleles (Sykes, Ogilvie and Wordsworth, 1985) to the more than 80 sites at D14S1 a (Balazs et al., 1986). 人的基因组中的高可变位点的总数有多少目前还不知道,但这个数字有可能很大。 The total number of human genome hypervariable loci how much is not known, but this figure may be large. 人的基因组可能含有至少1500个高可变区。 Human genome might contain at least 1500 hypervariable regions.

业已发现,有可能克隆一个通过将基因组DNA的片段同某多核苷酸探针杂交所鉴定的DNA片段,上述探针是能够通过参考一种以上的多态性小随体区或高可变位点鉴别DNA的多核苷酸探针,例如在前面所述的英国专利申请No.8525252(公开号2166445)中所描述并提出权利要求的探针33.6和33.15;并可以由其制备出能够杂交到一DNA片段上的多核苷酸或探针,该DNA片段含有一个对于基因组中的某特定区域或位点为特异的小随体,而所说的区域或位点是一个可以提供信息的基因位点。 It has been found, it is possible by cloning a fragment of genomic DNA hybridized with a polynucleotide probe identified DNA fragment, the probe is able to reference more than one polymorphic minisatellite region or hypervariable locus point evaluator DNA polynucleotide probes, for example in the aforementioned British Patent Application No.8525252 (Publication No. 2166445) as described and claimed in claim probes 33.6 and 33.15; and can be prepared therefrom is capable of hybridizing to polynucleotide or probe a DNA fragment on the DNA fragment containing a gene group for a particular region or locus-specific minisatellite for, and said region or locus is a gene locus can provide information points. 在本说明书中我们将提供信息的基因位点定义为:在任何100,个随机选取的无关个体的样品中的该位点处可以鉴别出至少3个不同的等位基因。 In the present specification, we will provide information locus defined as: in any 100, a sample of randomly selected unrelated individuals in the site can identify at least three different alleles. 本文中将这一含义表述为,该位点具有至少3(100)个等位基因变异。 In this article, the meaning of this expression is that the site has at least three (100) alleles. 随机选取的无关个体的样品数较好为500个,最好是1000个。 Number of samples randomly selected unrelated individuals is preferably 500, preferably 1000. 在本文中将在这样一些样品中区分出至少3个不同的等位基因的能力分别称为3(500)和3(1000)。 Will be used herein to distinguish at least 3 different alleles in the ability of some samples such are referred to 3 (500) and 3 (1000). 重要的是这100、500或1000个随机选取的无关个体的样品必须确实是随机选取的,因为通常情况下通过筛分总人群中的足够大数量的样本,可以鉴别100、500或1000个在给定的提供信息之基因位点上具有3个以下等位基因的个体。 It is important that 100,500 or 1000 randomly selected sample of unrelated individuals must really be randomly selected, because usually by screening the general population of a sufficiently large number of samples can be identified 100, 500 or 1000 in given below provide a three allele of the locus information.

在一个给定的提供信息的基因位点处,这种多态性越高,这种位点特异的探针在遗传特性鉴定中(例如为确定父亲身份或法医鉴定而进行个体鉴别时)就越有用并且提供更可靠的信息。 At a given locus at the information provided, the higher this polymorphism, this location-specific probe point in genetic characterization (eg, to determine paternity or forensic identification of individuals when performed) on The more useful and provide more reliable information.

在这方面,可将上述“个体”一词理解为不仅用来指人,而且还指动物和植物以及由这些人、动物和植物得到的细胞系。 In this regard, the above "individual" the term is understood to refer not only to human, but also animals and plants as well as by means of these people, animals, and plant-derived cell lines. 但是在每一种情况中,随机选取的无关个体的样品应该都是取自同一物种。 However, in each case, the sample of randomly selected unrelated individuals should all taken from the same species.

就本发明应用于人的情况而言,本文中所使用的“信息性基因位点”这一表述可以换一种方式定义为:一个基因位点,在该位点处在从选自下列任何20个细胞系〔这些细胞系已经寄存在美国典型培养物保藏中心(ATCC)〕中提取的DNA中可以区分出至少3个不同的等位基因。 The present invention is applied to the human situation, the "informative genetic locus" as used herein, the expression can be defined as another way: a gene locus, the locus is selected from any of the following in 20 cell lines [these cell lines have been deposited with the American Type Culture Collection (ATCC)] in the extracted DNA can distinguish at least 3 different alleles.

细胞系 ATCC寄存号Hela CCL2RPMI2650 CCL30Detroit532 CCL54Detroit525 CCL65Detroit529 CCL66Detroit510 CCL72WI-38 CCL75Citrullinemia CCL76EB-3 CCL85RAJI CCL83JIYOYE(P-2003) CCL87 Cell line ATCC deposit No. Hela CCL2RPMI2650 CCL30Detroit532 CCL54Detroit525 CCL65Detroit529 CCL66Detroit510 CCL72WI-38 CCL75Citrullinemia CCL76EB-3 CCL85RAJI CCL83JIYOYE (P-2003) CCL87

WI-26 CCL95Detroit551 CCL110RPMI6666 CCL113RPMI7666 CCL114CCRF-CEM CCL119CCRF-SB CCL120HT-1080 CCL121HG261 CCL122CHP3(MW) CCL132LL47(MaDo) CCL135HEL299 CCL137LL24 CCL151HFLI CCL153WI-1003 CCL154MRC-5 CCL171IMR-90 CCL186LS174T CCL188LL86(LeSa) CCL190LL97A(AIMy) CCL191HLF-a CCL199CCD-13Lu CCL200CCD-8Lu CCL201CCD-11Lu CCL202CCD-14Br CCL203 WI-26 CCL95Detroit551 CCL110RPMI6666 CCL113RPMI7666 CCL114CCRF-CEM CCL119CCRF-SB CCL120HT-1080 CCL121HG261 CCL122CHP3 (MW) CCL132LL47 (MaDo) CCL135HEL299 CCL137LL24 CCL151HFLI CCL153WI-1003 CCL154MRC-5 CCL171IMR-90 CCL186LS174T CCL188LL86 (LeSa) CCL190LL97A (AIMy) CCL191HLF-a CCL199CCD- 13Lu CCL200CCD-8Lu CCL201CCD-11Lu CCL202CCD-14Br CCL203

CCD-16Lu CCL204CCD-18Lu CCL205CCD-19Lu CCL210Hs888Lu CCL21MRC-9 CCL212Daudi CCL213CCD-25Lu CCL215SW403 CCL230NAMALWA CRL1432上述所有细胞系均可随意向ATCC索取,地址是:12301 Parklawn Drive,Rockville,Maryland 20852-1776,USA。 CCD-16Lu CCL204CCD-18Lu CCL205CCD-19Lu CCL210Hs888Lu CCL21MRC-9 CCL212Daudi CCL213CCD-25Lu CCL215SW403 CCL230NAMALWA CRL1432 all these cell lines can be freely obtained from the ATCC, the address is: 12301 Parklawn Drive, Rockville, Maryland 20852-1776, USA. 这些细胞系已列入ATCC的细胞系与杂交瘤的目录册中。 These cell lines have been included in ATCC hybridoma cell lines and in catalogs. 上述细胞系都是在1985年以前寄存在ATCC的。 These cell lines were previously registered in 1985 in the ATCC.

根据本发明的一个特征,其提供了一种制备能够杂交到一个DNA片段上的多核苷酸的方法,该DNA片段含有一个对于基因组中的某特定区域或位点是特异的小随体,所述的区域或位点具有至少3(100)个等位基因变异(根据上文中的定义)。 According to one feature of the present invention, there is provided a method of preparing a polynucleotide capable of hybridizing to a DNA fragment of the method, the DNA fragment containing a gene group for a particular region or locus is specific minisatellite, the said region or locus having at least 3 (100) alleles (according to the above definition). 该方法包括克隆一个通过将基因组DNA的片段与一多核苷酸探针杂交而鉴定的DNA片段,上述多核苷酸探针能够参考一个以上的多态性小随体区域高可变位点来鉴别DNA;由此制备能够杂交到一含小随体之DNA片段上的多核苷酸,所述的小随体对于基因组中的某特定区域或位点是特异的,而所述的区域或位点具有至少为3(100)个等位基因变异(根据上文中定义)。 The method comprises cloning a fragment of genomic DNA by the hybridization with a polynucleotide probe to identify the DNA fragments and the polynucleotide probes can refer to more than one polymorphic minisatellite region hypervariable loci to Identification of DNA; thus prepared is capable of hybridizing to a DNA fragment containing the polynucleotide of the minisatellite of the minisatellite genome for a particular region or locus is specific, and said region or locus point having at least 3 (100) alleles (according to the definition above).

根据本发明的另一特征,其提供了一种制备能够杂交到一DNA片段上的多核苷酸的方法,该DNA片段含有一个对于基因组中的一个特定区域或位点是特异的小随体,而所述的区域或位点的多态性程度至少为3(根据上文定义)。 According to another feature of the present invention, there is provided a method of preparing a polynucleotide capable of hybridizing to a DNA fragment of the method, the DNA fragment containing a gene group for a particular region or locus is specific minisatellite, The polymorphic region or site of the extent of at least 3 (as defined above).

这一方法包括克隆一个通过将基因组DNA的片段与一个多核苷酸探针杂交而鉴定的DNA片段(该探针能够参考一种以上多形性小随体区域或高可变位点鉴定DNA);由此制备能杂交到一个含小随体之DNA片段上的多核苷酸,该小随体对于基因组中的一个特定区域或位点是特异的,而所述的区域或位点具有的多态性程度至少为3(根据上文定义)。 This method comprises cloning a fragment of genomic DNA by a polynucleotide probe and hybridization to identify a DNA fragment (the probe can reference more than one polymorphic minisatellite region or hypervariable locus identified DNA) ; thus prepared are capable of hybridizing to a polynucleotide containing a minisatellite DNA fragments on the body of the minisatellite genome for a particular region or locus is specific, and said region or locus having multiple degree at least three states (according to the definition above).

多核苷酸探针最好包含同时包括有己标记或标志组件的、至少含有3个串联重复(在所有串联重复的序列之间平均至少有70%的同源性)序列的多核苷酸,这些序列与基因组的小随体区是同源的,其相符程度为能使探针杂交到用限制性核酸内切酶切断样品DNA所得到的一个相应的DNA片段上,其中:a)每次重复都含有一个核心,该核心与来自不同之基因位点的多个小随体中存在的有相似长度的相符核心区至少有70%的同源性(即70%相符); Polynucleotide probe preferably contains both include hexyl mark or sign assembly, comprising at least three tandem repeats (between all tandemly repeated sequences of an average of at least 70% homology) polynucleotide sequences, these sequences with a minisatellite region of the genome are homologous, its conformity to enable probe hybridizes to a nucleic acid with a restriction endonuclease cutting a corresponding DNA fragment obtained DNA samples, wherein: a) each repetition comprising a core, the core with a plurality of small differences from the minisatellite loci have similar length present in the core area is consistent with at least 70% homology (or 70% of the time);

b)这一核心长度为6至16个核苷酸; b) the core length from 6 to 16 nucleotides;

c)对该核心没有贡献的重复单元中核苷酸的总数不超过15个。 c) the total number of the repeating units in the core does not contribute no more than 15 nucleotides.

另外,上述DNA片段含有一来自小随体区或高可变位点的小随体,这个区域或位点可以用一个多核苷酸探针测到,所述的多核苷酸探针包含序列(包括其互补序列)(A)AGGGCTGGAGG 1(其中T是T或U,(A)可有可无),或序列(包括其互补序列) Further, the DNA fragment containing a minisatellite from a minisatellite region or hypervariable locus, this region or locus can be measured to a polynucleotide probe, said polynucleotide probe comprising the sequence ( including its complementary sequence) (A) AGGGCTGGAGG 1 (wherein T is T or U, (A) optional), or the sequence (including complementary sequences)

AGAGGTGGGCAGGTGG 2(其中T是T或U)的至少三次串联重复。 AGAGGTGGGCAGGTGG 2 (wherein T is T or U) at least three tandem repeats.

根据本发明的另一特征,其提供了一个分离形式或已克隆形式的核苷酸,它包含一个对单一小随体区或高可变位点特异的核苷酸序列,并且是与之同源的,其相符程度为能使该核苷酸序列杂交到用限制性核酸内切酶切断样品基因组DNA所得到之相应的DNA片段上,所说的DNA片断含有一个来自所述之小随体区或高可变位点的小随体,其中:1)上述区域或位点具有至少为3(100)的等位基因变异(根据本文中的定义); According to another feature of the invention, which provides an isolated form or in the form of nucleotides has been cloned, which nucleotide sequence contains a single minisatellite region or hypervariable locus-specific, and is associated with source, which enables the degree of coincidence of the nucleotide sequence to hybridize with the restriction endonuclease cut genomic DNA samples were obtained on the corresponding DNA fragments, said DNA fragment containing a minisatellite from said of minisatellite region or hypervariable locus, wherein: 1) said region or locus having at least 3 (100) is an allelic variation (as herein defined);

2)上述区域或位点可以用一个多核苷酸探针检测到,该探针含有序列(包括互补序列):(A)AGGGGCTGGAGG 1(式中T是T或U,(A)可有可无),或者序列(包括互补序列):AGAGGTGGGCAGGTGG 2(式中T是T或U)的至少三个串联重复序列。 2) said region or locus can use a polynucleotide probe is detected, the probe comprising the sequence (including complementary sequences) :( A) AGGGGCTGGAGG 1 (wherein T is T or U, (A) optionally ), or the sequence (including complementary sequences): AGAGGTGGGCAGGTGG 2 (wherein T is T or U) at least three tandem repeats.

本发明还涉及相应的多核苷酸,其中正与至少3个等位基因变异相对,上述的区域或位点具有的多形性程度至少为3。 The present invention also relates to a corresponding polynucleotide which is at least three allelic variants opposite, the above-described region or locus having a degree of polymorphism of at least 3.

根据本发明的另一特征,本发明的多核苷酸及采用上文中限定的本发明之方法制备的多核苷酸是通过增殖含有己克隆之物质的微生物或直接合成来制备的。 According to another feature of the invention, the polynucleotide of the invention and the use of a polynucleotide as defined above process of the present invention is prepared by preparing a microbial proliferation or direct synthesis of the substance has been cloned.

本发明包括DNA、RNA的多核苷酸以及可杂交到DNA上的任何其它类型的多核苷酸。 The present invention comprises DNA, RNA and the polynucleotide can hybridize to a polynucleotide of any other type of DNA on. 以上限定的多核苷酸是未标记的,且可以是双股(ds)或单股(ss)形式的。 Polynucleotide as defined above is unlabeled, and may be double-stranded (ds) or single stranded (ss) form.

本发明包括用作探针的ss形式的标记的多核苷酸以及用以制备ss-探针的其标记的ds-前体。 Used as a probe of the present invention comprises a labeled form of ss polynucleotides and their labeled ds- ss- probes used to prepare a precursor.

因而,根据本发明的另一特征,其提供了一种多核苷酸探针,它包括上文中所限定的本发明的多核苷酸或用上文中指定的方法制备的具有一标记或标志组件的多核苷酸。 Thus, according to another feature of the invention, there is provided a polynucleotide probe comprising a polynucleotide of the invention as defined above or with a marker or flag with the component specified above prepared polynucleotides. 一般地说,至少该多核苷酸的核苷酸序列应是单股形式的,最好是探针由整体上为单股形式的多核苷酸所构成。 Generally, at least the nucleotide sequence of the polynucleotide is single-stranded form should, preferably by the probe as a whole in the form of a single strand polynucleotide composed.

根据本发明的另一特征,其提供了一种制备上文中所限定的多核苷酸探针的方法,该方法包括标记或标志上文中所限定的本发明的多核苷酸或用上文中限定的本发明之方法制备的多核苷酸。 According to another feature of the present invention, there is provided a method of polynucleotide probe as hereinbefore defined in the preparation, the method comprising the marker or markers in the present invention hereinbefore defined or a polynucleotide as defined above The method of the present invention for preparing polynucleotide.

本发明的多核苷酸探针最好是以任何常规方式进行32p-放射性同位素标记,但也可以用杂交技术领域中所熟知的其它方法进行放射性同位素标记,以得到35s-放射性同位素标记的探针。 The polynucleotide probes of the present invention is preferably carried out in any conventional manner 32p- radiolabelled, but may also be other hybridization methods well known in the art be radiolabelled to give 35s- radioisotope labeled probe . 它们也可以按DCWard等人的方法用生物素或类似的物质标记〔Proceedings of the 1981ICN-UCLA Symposium on Developmental Biology using Purified Genes held in Keystone,Colorado on March 15-20,1981Vol.XXIII 1981Pages 647-658 Academic Press;Editor Donald D.Brown et al or even enzyme-labelled by the method of ADBMalcolm et al,Abstracts of the604 th Biological Society Meeting,Cambridge,England(Meeting of 1 July 1983)〕。 They can also press DCWard et al process with biotin or a similar substance labeled [Proceedings of the 1981ICN-UCLA Symposium on Developmental Biology using Purified Genes held in Keystone, Colorado on March 15-20,1981Vol.XXIII 1981Pages 647-658 Academic Press; Editor Donald D.Brown et al or even enzyme-labelled by the method of ADBMalcolm et al, Abstracts of the604 th Biological Society Meeting, Cambridge, England (Meeting of 1 July 1983)].

根据本发明的另一特征,其提供了一种参考一个或多个对照物对基因组DNA的试验样品进行特性鉴定的方法,该方法包括:用一种或多种限制性酶切断样品DNA,该限制性酶不至于将相应于串联重复的序列切至任何相关程度;用一个多核苷酸或多核苷酸探针探查这些DNA片段,上述多核苷酸或多核苷酸探针含有一个对于单一的小随体区或高可变位点为特异的并且是同源的核苷酸序列,其相符程度足可使该核苷酸序列能杂交到含有来自上述单一小随体区或高可变位点之小随体的相应DNA片段上;检测已杂交的DNA片段;将已杂交的片段与一个或多个上述的对照物进行比较,其中:1)所述的小随体区或高可变位点具有至少为3(100)的等位基因变异; According to another feature of the invention, there is provided a reference to one or more controls for the test samples of genomic DNA were characterization method, the method comprising: using one or more restriction enzymes cut the sample DNA, the restriction enzyme not corresponding to the tandem repeat sequence cut to any relevant extent; with one polynucleotide or polynucleotide probe probe DNA fragments, said polynucleotide or polynucleotide probe comprising a single small for minisatellite region or hypervariable locus-specific and is homologous to a nucleotide sequence, which is consistent enough to make the degree of nucleotide sequence capable of hybridizing to a solution from said single minisatellite region or hypervariable locus The small DNA fragment with the corresponding body; detecting hybridized DNA fragment; the fragment has been hybridized with one or more of the above controls are compared, wherein: 1) said minisatellite region or hypervariable locus point having at least 3 (100) allelic variants;

2)所述的区域或位点可以用能够参考一个以上多态性小随体区或高可变位点以鉴定DNA的多核苷酸探针来检测。 2) said region or locus can be detected with reference to one or more polymorphisms can minisatellite region or hypervariable locus to identify DNA polynucleotide probes.

本发明还涉及对试验样品进行特性鉴定的相应方法,在该试验样品中上述区域或位点具有与至少3个等位基因变异相对的至少为3的多态性程度。 The present invention also relates to a corresponding method for characterization of the test sample, the test sample in the said region or locus having an allelic variation of at least 3 relative to the degree of polymorphism of at least 3.

本发明的方法最好是采用一种多核苷酸或按本发明之方法制备的多核苷酸,或者采用上文中限定的本发明的多核苷酸探针来实施。 The best method of the present invention is the use of a polynucleotide or a polynucleotide prepared by the process of this invention, or the use of a polynucleotide probe as hereinbefore defined to implement the present invention.

样品DNA最好是人的DNA。 Preferably the sample DNA is human DNA.

为了帮助读者理解,现就本说明书中的某些术语说明如下:高可变的人、动物或植物DNA的某个被识别的位点或位点上的一个区域,如果它是以许多不同的形式(例如在长度或顺序方面)出现,则称之为高可变的。 To aid the reader in understanding, the present specification will now certain terms are described below: hypervariable person, an area of a locus or loci identified animal or plant DNA on, if it is based on a number of different form (e.g., in terms of length or sequence) occurs, is called a high variable.

小随体由一个短的DNA序列串联重复构成的人、动物或植物DNA的一个区域。 Minisatellite DNA by a short tandem repeat sequence consisting of human, animal or plant of a region of DNA. 所有重复单元可能不一定完全相同。 All repeat units may not necessarily be identical.

多态性的个体与个体之间或者在染色体配对的已知个体之间表现有变异性的基因或其它DNA片段,称之为多态性的。 Or between the individual and the individual polymorphisms between paired chromosomes known manifestations of individual variability in gene or other DNA fragments, called polymorphisms.

重复或串联重复(序列)完全或不完全地顺序重复的多核苷酸序列。 Repeat or Tandem Repeat (Sequence) complete or incomplete sequentially repeating polynucleotide sequence. 通常一个被说成是串联重复的序列应重复至少3次。 Usually one is said to be tandemly repeated sequence should be repeated at least 3 times.

不完全重复(序列)就核苷酸的数目或种类而言,其不是严格重复,但可识别出某相符顺序(Consensus Sequence)的重复。 Imperfect repeats (sequences) on the number or types of nucleotides, its not exactly duplicate, but can be identified in a consistent sequence repeat (Consensus Sequence) of.

%同源性(%相符)在比较两个重复或串联重复序列A和B时,将A中的碱基对数目减去B中为与A一致而必须替换、增加或删去的碱基对数,然后表示以百分数表示,即得到百分同源性。 % Homology (% match) when comparing two repeat or tandem repeat sequences A and B, the number of base pairs in A minus B for the A consistent and must be replaced, added or deleted base pairs number, and then expressed as a percentage, that percentage get homology. 据此可知,ATGC和AGC这两个序列之间的百分同源性是75%。 Accordingly understood, ATGC and AGC percent homology between the two sequences is 75%.

相符核心(Consensus Core)(序列)在许多重复序列中,通常是在有2个或2个以上不同的小随体的重复单元中,可以被鉴定为最接近之匹配的序列。 Match core (Consensus Core) (sequence) in a number of repeat sequences, usually there are two or more than two different repeat units in the minisatellite, can be identified as the closest match of the sequence.

核苷酸(nt)和碱基对(bp)作为同义词使用。 Nucleotide (nt) and base pair (bp) used synonymously. 两者均可以指DNA或RNA。 Both can refer to DNA or RNA. 缩写字C、A、G和T习惯上指的是(脱氧)胞苷、(脱氧)腺苷、(脱氧)鸟苷以及脱氧胸苷或尿苷。 The abbreviations C, A, G and T on is used to refer (deoxy) cytidine, (deoxy) adenosine, (deoxy) guanosine, and deoxythymidine or uridine. 但是应当理解,缩写字A、C、G和T可包括能与一般的核苷酸即(脱氧)腺苷、(脱氧)胞苷、(脱氧)鸟苷、及脱氧胸苷或尿苷进行碱基配对的其它碱基修变的核苷酸。 It should be understood abbreviations A, C, G and T may comprise a nucleotide i.e. with general (deoxy) adenosine, (deoxy) cytidine, (deoxy) guanosine, and thymidine or uridine base Other base repair base paired nucleotides changed. 这样的碱基修变的核苷酸包括(脱氧)肌苷和(脱氧)氮杂鸟苷。 Such nucleotides include bases modifier of (deoxy) inosine and (deoxy) guanosine aza.

当本发明的多核苷酸或多核苷酸探针含有串联重复的序列时,该串联重复可以是完全重复的也可以是不完全重复的,或者是完全重复与不完全重复的混合体。 When a polynucleotide or polynucleotide probe of the present invention contains tandemly repeated sequence, the tandem repeats may be an exact duplicate of repetition may be incomplete, or completely duplicate was repeated incomplete mixture. 在探针序列中较好有至少3个重复,最好是有至少7个重复序列。 In the probe sequence is preferably at least three repeat, preferably at least 7 repeats.

本发明的探针可以单独地或与其它位点特异的探针结合起来用于那些使用探针之混合物或合剂(Cocktail)的一系列试验或单个试验中,它们可以用于下列领域:1.人的父系或母系检验。 Probes of the invention may be used alone or in combination for those using a probe of the mixture or mixture (Cocktail) of a single test or series of tests with other site-specific probe, which can be used in the following areas: 1. paternal or maternal test person.

2.在例如移民争执和继承纠纷中进行家族检验。 2. Perform testing in family disputes such as immigration and inheritance disputes.

3.孪生儿的接合性试验。 3. The child's twin zygosity test.

4.人的近交检验。 4. Human inbred test.

5.人的一般谱系分析。 5. Generally people pedigree analysis.

6.鉴别与人的遗传病有联系的位点,从而可以构建特异的探针以检查基因缺陷。 6. Identification and human genetic disease loci linked to specific probes can be constructed to check for genetic defects.

7.法医学,例如(a)为取自被强奸之受害者的精液样品打指纹印迹; 7. forensic science, such as (a) is taken from the rape victim was hit in the semen sample fingerprinting;

(b)将取自例如弄污的衣服上的血、毛发和精液样品打指纹印迹,(c)鉴别人的尸体,(d)鉴定其它法医组织样品例如皮肤的遗传特性。 (B) will be taken from the example of blood soiled clothes, hair and semen samples were playing fingerprinting, (c) identify the person's body, (d) other forensic identification of genetic traits such as skin tissue samples.

8.细胞嵌合性(Cell Chimaerism)研究,例如在骨髓移植后追踪供体对受体细胞的嵌合性。 8. The cell chimerism (Cell Chimaerism) studies, e.g., after bone marrow transplantation track donor chimeric receptor cells.

9.家畜品种和谱系的分析/证明。 9. Analysis of livestock breeds and pedigree / proof. (例如,这可以包括纯种动物的常规控制和检验,以及在涉及赛马和狗品种等的诉讼案件中检验谱系)。 (For example, this may include conventional purebred animal control and inspection, as well as in litigation involving test pedigree dog breeds, such as horse racing and in). 此外,还提供可能表明与经济上重要的遗传特性有联系的基因标志。 In addition, it also provides may indicate the importance of the genetic characteristics of the economy have genetic markers linked.

10.经过培养的动物或植物细胞系的常规质量控制,核查纯细胞系的污染物以及常规鉴定工作。 10. After cultured animal or plant cell lines for routine quality control of conventional pollutants and appraisal verification pure cell lines.

11.分析肿瘤细胞及因分子异常而产生的肿瘤。 11. Analysis of tumor cells and tumor arising due to molecular abnormalities.

12.可以予料,由此得到的多核苷酸或探针在植物育种中具有潜在的应用前景。 12. can be expected, the polynucleotide or probe thus obtained has potential applications in plant breeding. 但是,如下文中实施例3所述,本发明的位点特异性探针在法医学中特别有用,且至少pλg3探针可以与胎儿血红蛋白之遗传稳定性的异源细胞形式共分离。 However, as described in Example 3, locus-specific probe of the present invention is particularly useful in forensic science, and at least pλg3 probe can be in the form of co-segregated with the heterologous genetic stability of the cells of fetal hemoglobin. 这种探针还提供了在父系检验和有关方面的用途(见上述用途2和5)以及作为细胞嵌合性研究的有效方法。 This probe also provides testing and use in paternal interested parties (see above uses 2 and 5), and as an effective method of chimerism studies.

图1A显示取自DNA指纹图谱的一个特定高可变DNA片段的凝胶电泳图谱,这个片段在本文中称为“g片段”。 Figure 1A shows a particular DNA fingerprint from the hypervariable gel electrophoresis of DNA fragments, the fragments called "g fragment" in this article. 图1B借助克隆DNA的限制图显示出DNA g片段的组织。 With the cloning of DNA Figure 1B shows the restriction map of DNA g tissue fragments.

图2显示高可变之g片段的DNA序列,特别着重强调了相符重复序列及其与英国专利公开NO2166445中“核心”序列的校准排列部分。 Figure 2 shows the DNA sequence of the hypervariable segment g, particularly highlighted the match repeats its arrangement with the British patent discloses calibration NO2166445 part in the "core" sequence.

图3显示用pλg3检测到的多态性DNA片段的孟德尔(Mendelian)遗传特征; Figure 3 shows the Mendelian genetic characteristics detected by pλg3 polymorphic DNA fragments (Mendelian);

图4显示通过杂交到pλg3上检测到的小随体等位基因的大小分布; Figure 4 shows on pλg3 detected by hybridization minisatellite allele size distribution;

图5显示用于位点特异性高可变DNA探针的λMS重组体的筛分; Figure 5 shows for the site-specific hypervariable λMS recombinant screening DNA probes;

图6显示λMS系列小随体和pλg3小随体的相符重复序列单元; Figure 6 shows λMS series minisatellite and pλg3 small body with consistent repeat unit;

图7显示用本文中称为λMS32的探针检测到的高可变位点的孟德尔(Mendelian)遗传特征; Figure 7 shows a probe with Mendel referred to herein as λMS32 detected hypervariable loci (Mendelian) genetic characteristics;

图8显示对用本文中称为λMS43的探针检测到的高可变位点处等位基因变异性的估计; Figure 8 shows an estimate of the use of the probe referred to herein as λMS43 detected variability at the hypervariable locus allele;

图9显示对人的DNA库中高可变位点处等位基因长度变异的分析; Figure 9 shows a library of human DNA at hypervariable loci analysis of allelic length variation;

图10显示在一组用AluⅠ酶切的人-啮齿动物体细胞杂交DNA中,用本文中称作λMS32的探针检测到的高可变位点的等位基因分离; Figure 10 shows a set of use AluⅠ digested human - rodent somatic cell hybrid DNA, the use of the probe referred to herein as λMS32 detected alleles isolated hypervariable locus;

图11显示对位点特异性高可变探针之灵敏度的估价及其在强奸/杀人双重犯罪案件的法医学分析中的应用; Figure 11 shows the variable of locus-specific probes of high sensitivity of the valuation and the rape / murder dual forensic analysis of criminal cases in the application;

图12显示,通过与本发明之集合的小随体探针杂交,检测人DNA中的多个高可变位点。 Figure 12 shows, by hybridization with minisatellite probe set of the present invention, a plurality of detection of human DNA in the hypervariable loci.

本发明的多核苷酸、用本发明的方法制备的多核苷酸以及本发明的多核苷酸探针可以同含有一来自小随体区或高可变位点之小随体的DNA片段杂交,上述区域或位点可以用一多核苷酸探针鉴别出来,该探针是由选自下列的任一序列:AGGAATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6 Polynucleotide of the present invention, polynucleotides prepared by the method of the present invention and the polynucleotide probe with the present invention may contain a small minisatellite region or hypervariable locus of the body from the hybridization with the DNA fragment, said region or site may be more than one polynucleotide probe identification out of the probe is selected from any one of the following sequences: AGGAATAGAAAGGCGGGYGGTGTGGGCAGGGAGRGGC 3GTGGAYAGG 4TGGGAGGTGGRYAGTGTCTG 5GAATGGAGCAGGYGRCCAGGGGTGACTCA 6

GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TCTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9(其中Y是C、T或U,X是G或C,R是A或G,T是T或U),或它们的串联重复序列、或与它们互补的序列所构成的。 GGGCTGGGGAGATGGTGGAGGAGGTGTTGG 7AGGCTGGGGAGATGGTGGAGGAAGAGTAC 8TCTGTGTAATGGGTATAGGGAGGGCCCCGGGAAGGGGGTGTGGYX 9 (wherein Y is C, T or U, X is G or C, R is A or G, T is T or U), or tandem repeats thereof or a sequence complementary with them posed.

本发明的多核苷酸、用本发明的方法制备的多核苷酸以及本发明的探针均能与含有一个来自单一特定小随体区或高可变位点之小随体的DNA片段杂交,因而是位点特异的。 Polynucleotide of the present invention, polynucleotides and probes of the invention by the method of the present invention is prepared from a can containing a single specific minisatellite region or hypervariable locus and small DNA fragments with the body of hybridization, Thus is site-specific. 一般地说,本发明的多核苷酸和探针在非常严格的杂交条件下是位点特异的。 In general, polynucleotides and probes of the invention under highly stringent hybridization conditions are site-specific. 在这方面,本文中使用“位点特异的”一词来表述多核苷酸或探针意思是该多核苷酸或探针在确保多核苷酸或探针只杂交到单一特定位点的杂交条件下才能使用。 In this regard, as used herein, the term "site-specific" to express a polynucleotide or probe means that the polynucleotide or probe polynucleotide or to ensure that only probes hybridized to a single specific site of hybridization conditions next to use.

但是应当理解到,在严格程度降低的杂交条件下使用时,本发明的位点特异探针可以参考一个以上多态性小随体区或高可变位点来鉴别DNA,因而可以用于鉴定其它提供信息的基因位点。 It should be understood that, when used under hybridization conditions of reduced stringency, site-specific probe of the present invention can refer to more than one polymorphic minisatellite region or hypervariable locus to identify DNA, which can be used to identify Other loci provide information.

从而,根据本发明的另一特征,其提供了制备能杂交到一个含有小随体之DNA片段上的多核苷酸的方法,该小随体对于基因组中的一个特定区域或位点是特异的,所述的区域或位点具有至少3(100)个等位基因变异(根据本文中的定义)。 Thus, according to another feature of the present invention, there is provided a process for preparing a polynucleotide capable of hybridizing to DNA fragments of the method of the minisatellite containing the minisatellite genome for a particular region or locus is specific for , said region or locus having at least 3 (100) alleles (according to the definition herein). 这一方法包括:将基因组DNA的片段与上文中限定的多核苷酸探针杂交,克隆该通过杂交鉴定的DNA片段,由其制备能杂交到含有小随体之DNA片段上的多核苷酸,该小随体对于基因组中的一个特定区域或位点是特异的,所述的区域或位点具有至少3(100)个等位基因变异(根据本文中的定义);该方法应在使探针可以参考一个以上多态性小随体区域或高可变位点鉴别DNA的杂交条件下有效地实施。 This method comprises: the polynucleotide probe hybridization of genomic DNA fragments hereinabove defined, clones identified by hybridization to the DNA fragment prepared therefrom are capable of hybridizing to a DNA fragment containing the polynucleotide of minisatellite on, The minisatellite genome for a particular region or locus is specific, said region or locus having at least 3 (100) an allelic variation (as herein defined); the method should make EXPLORATION needle can refer to hybridization conditions or hypervariable regions of DNA loci to identify a more effective implementation of polymorphic minisatellite.

根据本发明的另一特征,其提供了一种分离形式或已克隆形式的多核苷酸,它包含一个对单一小随体区或高可变位点为特异的核苷酸序列,并且该序列与之同源,其相符程度达到可使该核苷酸序列能杂交到用限制性核酸内切酶切断样品基因组DNA得到之相应的DNA片段上,所说的DNA片段含有一个来自所述小随体区或高可变位点的小随体,其中:1)所述的区域或位点具有至少3(100)个等位基因变异(根据本文中定义); According to another feature of the invention, which provides an isolated form or cloned forms of the polynucleotide, which contains a single minisatellite region or hypervariable locus for specific nucleotide sequence, and the sequence homologous thereto, which enables the degree of coincidence reach capable of hybridizing to the nucleotide sequence with the restriction endonuclease cut to give a sample of genomic DNA of the corresponding DNA fragments, said DNA fragment containing a minisatellite from said body minisatellite region or hypervariable locus, wherein: 1) said region or locus having at least 3 (100) an allelic variation (as defined herein) according to;

2)所述的区域或位点可以用一个上文中限定的位点特异性多核苷酸探针检测。 2) said region or locus can be defined hereinabove in a locus specific polynucleotide probe assay.

本发明还涉及相应的方法及相应的多核苷酸,其中所述的区域或位点具有至少为3的多态性程度。 The present invention also relates to a corresponding method and a corresponding polynucleotide, wherein said region or locus having a degree of polymorphism of at least 3.

本发明还涉及包含这样的位点特异性多核苷酸的探针以及用这样的多核苷酸及多核苷酸探针对DNA试验样品作遗传特性鉴定的方法。 The present invention further relates to probes comprising such locus specific polynucleotides and methods of genetic characterization of the use of such polynucleotides and polynucleotide probes for the test sample of DNA.

本发明的位点特异性多核苷酸和探针可以与含小随体之DNA片段的小随体是相似的,或者是与小随体之侧翼序列的相似性较差。 Site of the present invention specifically polynucleotides and probes may minisatellite DNA fragments containing the body of the minisatellite are similar, or with a small similarity with the body of the flanking sequence is poor.

在本发明的多核苷酸和探针包含一个特定序列的串联重复时,这样的序列一般应重复至少3次,并且这些重复不一定必须是严格的重复。 When the series polynucleotides and probes of the present invention comprise a specific sequence repeat, such sequences should generally be repeated at least three times, and these need not be repeated strict repetition. 不是严格的重复在其它地方称为不完全重复。 Not strictly repeated elsewhere called incomplete duplication. 不过这些重复应该可以看出来是被重复了的。 However, these should be seen repeated be repeated in. 一般地说,这意味着在所有重复单元之间平均有至少70%的同源性(相符性)。 In general, this means that an average of at least 70% homology (Conformance) between all repeating units. 在全部重复单元之间,较好有至少80%,更好有至少85%,最好有90%的相符。 Between all repeating units, preferably at least 80%, more preferably at least 85%, preferably with 90% match. 然而可以理解到的是,在有些情况下,核苷酸序列或“重复单元”並不需要完完全全地重复。 It is to be appreciated that in some cases, the nucleotide sequence or "repeat unit" need not be completely repeated.

重复单元的数目n至少是5,但最好是至少10。 The number n of repeating units is at least 5, but preferably at least 10. 适合的n是10至40,但原则上n可以是任何数,甚至是由1至10000。 Suitable n is 10-40, but in principle n can be any number, even from 1 to 10,000.

在本发明的多核苷酸和探针包含一特定序列的串联重复时,任何位于侧翼的序列基本上是不相干的。 When connected in series, and the polynucleotide probes of the present invention comprise a specific sequence repeat, any flanking sequences are substantially irrelevant. 它们可被删除或者可以以任何数的核苷酸存在,例如可多达50000个,不过用这样长的探针去完成工作可能不如通常长度那样敏感。 They may be deleted or may be present in any number of nucleotides, e.g., up to 50,000, but with such a long probe to complete the work may not be as sensitive as usual length. 然而象这样大的多核苷酸仍可用作载体,可由其中切下用作探针的较小的多核苷酸。 However, as such large polynucleotides still serve as carriers, can be used as a probe in which the cut smaller polynucleotides. 甚至当重复序列是ss-DNA形式时,这些位于侧翼的序列可以构成ds-DNA或ss-DNA的一个部分。 Even when the repeat sequences are ss-DNA form, these flanking sequences may form part of the ds-DNA or ss-DNA of.

本发明的多核苷酸和多核苷酸探针最好包含由上文中限定的序列式3至9中选出的某一序列或其互补序列,或者是其串联重复(在所有串联重复序列之间,平均有至少70%相符)或与之互补的序列。 Polynucleotide of the invention and polynucleotide probes preferably comprise a sequence defined by the formula above, or a sequence complementary to the sequence selected from 3-9, or tandem repeat (in between all tandemly repeated sequences , an average of at least 70% of the time) or a sequence complementary thereto.

本发明的一个优选的多核苷酸包含如上文所限定的序列式3的序列,或者其串联重复或与之互补的序列。 A preferred embodiment of the invention the polynucleotide comprises a sequence as hereinbefore defined sequence of formula 3, or tandem repeats thereof or a sequence complementary thereto. 这样的核苷酸序列是对位于染色体7上的常染色体的位点特异的。 Such a nucleotide sequence is an autosomal locus located on chromosome 7-specific. 包含这样一个序列的多核苷酸和多核苷酸探针在本文中被称为pλg3。 Polynucleotides and polynucleotide probes comprising such a sequence are referred to herein pλg3.

本发明的另一优选的多核苷酸包含如上文所限定的序列式4的序列,或者其串联重复或与之互补的序列。 Another preferred polynucleotide sequences of the present invention as hereinbefore defined comprising a sequence of formula 4, or tandem repeats thereof or a sequence complementary thereto. 这样的核苷酸序列是对位于染色体1上的位点特异的。 Such a nucleotide sequence is specific locus located on chromosome 1 on. 包含这样一个序列的多核苷酸或多核苷酸探针在本文中被称为λMS1。 A polynucleotide or polynucleotide probe comprises a sequence is referred to herein as λMS1.

本发明的另一优选的多核苷酸包含如上文所限定的序列式5的序列,或者其串联重复或与之互补的序列。 Another preferred polynucleotide of the present invention comprises the sequence of formula 5 as hereinbefore defined or tandem repeats thereof or a sequence complementary thereto. 这样的核苷酸序列对位于染色体7上的位点是特异的。 Such a nucleotide sequence of the locus is located on chromosome 7 is specific. 包含这样一个序列的多核苷酸和多核苷酸探针在本文中称为λMS31。 Polynucleotides and polynucleotide probes comprising such a sequence are referred to herein as λMS31.

本发明的另一优选的多核苷酸包含如上文所限定的序列式6的序列,或其串联重复或与之互补的序列。 Another preferred polynucleotide sequences of the present invention as hereinbefore defined comprising a sequence of formula 6, or a tandem repeat or a sequence complementary thereto. 这样的核苷酸序列是对位于染色体1上的位点特异的,包含这样一个序列的多核苷酸和多核苷酸探针在本文中被称为λMS32。 Such a nucleotide sequence is located on chromosome 1 on a site-specific, comprising a sequence of such polynucleotides and polynucleotide probe is referred to herein as λMS32.

本发明的另一优选的多核苷酸包含如上文所限定的序列式7和8中选出的序列,或者是其串联重复或与之互补的序列。 Another preferred polynucleotide sequences of the present invention of formula 7 and 8 comprises a sequence selected from as hereinbefore defined, or tandem repeats thereof or a sequence complementary thereto. 这样的核苷酸序列对位于染色体5上的位点是特异的。 Such a nucleotide sequence of the locus is located on chromosome 5 is specific. 包含这样一个序列的多核苷酸和多核苷酸探针在本文中被称为λMS8。 Polynucleotides and polynucleotide probes comprising such a sequence are referred to herein λMS8.

本发明的另一优选的多核苷酸包含如上文所限定的序列式9的序列,或者是其串联重复或与之互补的序列。 Another preferred polynucleotide sequences of the present invention as hereinbefore defined comprising a sequence of formula 9, or tandem repeats thereof or a sequence complementary thereto. 这样的核苷酸序列是对位于染色体12上的位点特异的。 Such a nucleotide sequence is located on chromosome 12 specific loci on. 包含这样一个序列的多核苷酸和多核苷酸探针在本文中被称为λMS43。 Polynucleotides and polynucleotide probes comprising such a sequence are referred to herein λMS43.

如上所述,在重复单元之间通常有至少70%,较好是至少80%,更好是有至少85%,最好是有至少90%的相符。 As described above, between the repeating units generally have at least 70%, preferably at least 80%, more preferably at least 85%, preferably at least 90% have a match. 但特别优选的是序列式3至9中任一序列的准确重复。 However, particularly preferred are any one of the exact sequence of the sequence of Formula 3-9 was repeated. 关于这一点应理解到的是,只要每个重复单元具有落入预期之序列式范围内的序列,即可使后一要求得到满足。 In this regard should be understood that, as long as each of the repeating unit having the sequence of sequential fall within the expected range, to make the latter requirement is met. 因而並不需要每个重复单元都具有完全相同的序列。 Thus each repeating unit does not need to have exactly the same sequence.

在本发明的一个优选的实施方案中,其提供了一个鉴定试验样品供体与一个或多个对照试验样品供体是完全一致或是不同的方法。 In a preferred embodiment of the present invention, which provides an identification of the test sample donor control test sample donor is identical or different, with one or more methods. 在该方法中,对照样品均须作彼此类似地酶切,並将来自各不同样品的杂交片段进行比较。 In this method, each control sample must be similarly digested, and the hybridizing fragments from various samples were compared.

在本发明的另一优选实施方案中,提供了一种鉴定试验样品供体与一个或多个对照样品供体之间家族关联的方法,在该方法中,对照样品均须作相似地酶切,並且就得自各个样品的杂交片段进行比较。 In another preferred embodiment of the present invention, there is provided a method for identifying a test sample between family donor and one or more control sample donors associated, in this method, the control sample shall be similarly digested , and have self-hybridizing fragments of each sample were compared.

可使用本发明的多核苷酸或探针实施上述方法,或可使用至少两种本发明的多核苷酸或探针,完成一系列或单个使用上述多核苷酸或探针之混合物的试验。 The invention may be used to implement the above polynucleotide or a probe method, or use of at least two polynucleotides or probes of the present invention, the completion of a series or a single use of the test mixture of the above-described polynucleotides or probes.

在本发明的另一优选实施方案中,其提供了一种诊断遗传性疾病、异常或特征的方法,该方法包括使用至少一个如上文所限定的多核苷酸或多核苷酸探针以检测试验样品,所述的探针是与特殊的遗传性疾病,异常或特征有关联的。 In another preferred embodiment of the present invention, there is provided a method for diagnosis of genetic disease, disorder or features, the method comprises using at least one as defined above polynucleotides or polynucleotide probes to detect test sample, said probe is specific genetic disease, disorder or features associated.

我们已于严格的杂交条件下,试验了所有用作位点特异性探针的本发明的探针。 We have under stringent hybridization conditions, all test probes used locus-specific probes of the present invention.

依据我们已试验过的本发明的探针检测的位点之极端特异性,並结合它们在Southern印迹杂交中显示的灵敏性,其在个体鉴别中,特别是当如英国专利申请NO.8525252(公告号为216644)中所述及並提出权利要求的可用于多位点DNA指纹分析之DNA不够用时,本发明便提供一种特别有用的试验工具。 Based on the extreme point of the probe to detect specific sites we have tested the present invention, and they appear in the combination of Southern blot hybridization sensitivity, which in individual identification, particularly when e.g. British Patent Application NO.8525252 ( Bulletin No. 216 644) as described and claimed and can be used for multi-locus DNA DNA fingerprinting analysis of the time is not enough, the present invention will provide a particularly useful test tool. 多位点DNA指纹图可由0.5μg DNA分析得到,而位点特异性探针则至少更敏感一个数量级,並且使用少至1μl的血液样品即可。 Multi-locus DNA fingerprints obtained by 0.5μg DNA analysis, and site-specific probe is at least an order of magnitude more sensitive, and use as little 1μl blood sample can be.

在需要检验甚至更少量的样品的任何非寻常情况下,将本发明的方法与欧洲专利申请NO86302299.2(公告号为0201184)中所述的放大技术相结合,将会具有特别显著的应用价值。 In the need to verify any unusual circumstances even a smaller amount of sample, the method of the present invention with the European Patent Application NO86302299.2 (Publication No. 0201184) amplification technique described in combination, will have a particularly significant value .

本发明能够在人的基因组中鉴定出可提供遗传信息的基因位点,並因而能够制备位点特异性探针;而每个位点特异性探针对单一之信息性基因位点又是特异的。 The present invention can be in the human genome identified genetic loci may provide information, and thus can be prepared by locus-specific probes; each locus-specific probes for the single informative genetic locus is specific a. 我们已特别鉴定了6个不同的信息性基因位点,这些位点在被检人群中具有很高的杂合性(至少90%),並且是迄今已分离到的基因位点中最具多态性的;我们也已制备了适用于每个位点的位点特异性探针,並将其分别定名为pλg3、λMS1、λMS8、λMS31、λMS32和λMS43(如上文所规定的)。 We have identified six different special informative loci, these loci with high heterozygosity (at least 90%) were seized in the crowd, and is so far isolated the most multi-loci normality; we also have prepared suitable sites for each locus-specific probes, and were named pλg3, λMS1, λMS8, λMS31, λMS32 and λMS43 (as defined above).

用位点特异性探针检测基因型的个体特异性程度可由每个位点处的异质性H来估计。 With locus-specific probes detected by a degree of specificity genotype at each locus heterogeneity H estimated. 一个位点处的平均等位基因频率q可由式(1-H)求出,而且两个随机选择的个体分享同一基因型的概率是q2(2-q)。 Mean allele frequency q at a locus by the formula (1-H) is obtained, and the two randomly selected individuals share the same genotype is the probability of q2 (2-q). 这种两个个体间假性关联的概率变异可由λMS8的0.02到λMS31的0.0002,而且因q中的异质性将会减少这些概率数,故这只是一个保守的估计。 This probability of false association between two individuals by λMS8 variation of 0.02 to 0.0002 λMS31, but also because of the heterogeneity of q will reduce the number of these probabilities, so this is a conservative estimate. 所有6个小随体探针的假性关联之累积概率是~10-16,差不多与使用1个多位点探针检测的两个随机选择的DNA指纹图之差数相同(见英国专利申请NO.8525252)。 The cumulative probability of false association with all six small probe body is ~ 10-16, the difference between two similar points using a probe to detect a number of randomly selected DNA fingerprints of the same number (see British Patent Application NO.8525252). 相反,就一个给定的高可变位点来说,两个血亲完全相同之或然率可由公式1/4(1+q)2得出。 On the contrary, it is a given hypervariable locus, the probability of two identical blood by Equation 1/4 (1 + q) 2 derived. 对于所有6个探针,血亲关系完全相同之累积概率为0.0004,此可与使用一个多位点探针所测得的DNA指纹图的~10-7相比较。 For all six probes, identical to the genetic relationship between the cumulative probability is 0.0004, this can be used with a multilocus probe measured DNA fingerprints of ~ 10-7 compared.

正如图11C中分析之谋杀案例所显示的,其中使用了由两个用于DNA指纹图分析的法医样品中回收到不足量的DNA,证明这些位点特异性高可变探针在法医学中是特别有用的。 As the murder case of FIG. 11C analysis of the show, which uses recycled by two forensic samples for DNA fingerprinting analysis of the insufficient amount of DNA, to prove that these site-specific hypervariable probe is in forensic science particularly useful. 用探针λMS1和λMS31检测显示有共同基因型的得自两受害者的精液DNA,不同于采自嫌疑犯的精液DNA基因型。 ΛMS1 λMS31 detection probe and display a common genotype DNA from semen of two victims, unlike the semen collected from the suspect DNA genotypes. 根据上述讨论,我们可以计算出受害者X和Y被不同的无关者强奸的概率为2×10-7。 Based on the above discussion, we can calculate the probability that X and Y are different victims of rape were unrelated to 2 × 10-7. 相似地,两个假定的强奸者是一级相关的,例如是兄弟的或然率为0.07。 Similarly, two hypothetical rape is a relevant example is the brother of probability is 0.07. 这就为X和Y是被同一个人强奸的提供了强有力的证据,后来通过对较大量法医材料的DNA指纹图分析进一步证实了这一结论。 This is X and Y are the same person raped provides strong evidence later by larger amounts of forensic material for DNA fingerprinting analysis further confirmed this conclusion. 再者,由X和Y取得的精液污物几乎肯定是来自于一个人,而不是两个或多个性攻击者。 Furthermore, the X and Y sperm get dirt almost certainly come from a person, rather than two or more personality attacker. 细察图11C中污斑b的实例,它显示出两个受害者等位基因和两个另外的强奸者等位基因。 Scrutinize instances stain b in Figure 11C, which shows two alleles and two other victims of rape who allele. 三个个体(两个强奸者和一个受害者)的储集物会显示4个或4个以下可分辨之等位基因的或然率可由公式q2(55-210q+216q2)求出。 Three individuals (two rapist and a victim) of petrophysical will display four or four of the following may be distinguished by the formula probability of allele q2 (55-210q + 216q2) is obtained. 由探针MS1的这一概率为0.02和由探针λMS31的这一概率为0.005,得出X被两个人而不是一个人强奸的联合概率为10-4。 The probability of a probe by MS1 is 0.02 and the joint probability by the probability of the probe λMS31 0.005, draw X by two people instead of one person rape 10-4. 这就确实了X和Y是被一个人强奸的,並且显示位点特异性探针-与多位点DNA指纹图探针不同-可用于估计在一混合的DNA样品中所呈现的个体数。 This is indeed the X and Y is a man of rape, and display site-specific probe - and multi-locus DNA fingerprinting probe different - the number of individuals in a mixed DNA samples can be used to estimate presented.

高可变DNA片段的蒙德尔遗传特征也可使之用于确定例如父权争异等家族关系问题。 Meng Deer hypervariable DNA fragments of genetic characteristics can also be used to determine, for example, such as the patriarchal family relations dispute different. 对于一个给定的位点特异性探针,孩子中的父系等位基因偶然存在于某个随机选择者中的概率可由2q-q2来估算。 For a given locus-specific probes, occasional children's paternal allele is present in a randomly selected person in the probability can be estimated 2q-q2. 对于所有六个小随体,得出非父亲之假性结论的累积概率为 For all six minisatellites, the cumulative probability of non-draw false conclusions for Father

,此与使用两个DNA指纹图探针获得的概率数差不多。 This and the use of two DNA fingerprint probes obtained a probability almost. 相似地,作出真正父亲的一级相关者(如兄弟)之结论的累积概率是0.02。 Similarly, the cumulative probability of making a real father's primary stakeholders (as brothers) the conclusion is 0.02. 因此依次使这些高可变位点特异性探针为确认有终止诉讼手续之申请的家族关系提供了一种十分有用的方法;但有些情况下,可因在这些不稳定的位点上突变成新的长度之等位基因,而导致错误的结论。 So in order to make these hypervariable loci specific probes confirmed the termination of the application for the proceedings of the family relationship procedures provide a very useful way; but in some cases, may be due to mutations in these unstable sites into a new allele length, and lead to erroneous conclusions. 有关这些高可变位点处的突变率尚不明瞭。 About these hypervariable loci at the mutation rate is not yet clear.

这些位点特异性探针的位点特异性和敏感性可使合并的探针用于产生多位点Southern印迹图谱(图12)。 Site specificity and sensitivity of these locus-specific probes enable pooled probes used to generate multi-locus Southern blot patterns (Fig. 12). 由5个合并的探针产生的这些“重建的DNA指纹图”,要比用多位点探针所检测的其对应图谱简单得多,而且会更适于在密度计扫描之后进行数字编码。 From 5 pooled probes produced these "reconstructed DNA fingerprint" than with the number of points corresponding to the detected probe spectrum is much simpler and more suitable for scanning densitometer after digitally encoded. 尽管个体间的小随体有很高的变异性,但还有相当数目(18%)的片段是在无关北欧人之间共有的,这主要是因为来自不同位点之小随体片段电泳共迁移的结果。 Although minisatellites have high inter-individual variability, but there are a significant number (18%) of the segment is shared between unrelated Nordic, mainly because of the different sites from small fragments with a total electrophoresis Migration results. 这一水平的泳带共享相似于由多位点探针产生的常规DNA指纹图中所见到泳带共享水平,后者在随机选择的个体间泳带共享的水平为~25%。 Swimming with shared this level is similar to conventional DNA fingerprinting probes generated from a number of points with a shared swimming in the level seen among individuals who were randomly selected with shared swimming level of ~ 25%. 因为在混合的探针图形中分辨出平均8.4个DNA片段,所以在一个个体中的所有这些片段存在于第二个随机选择的个体中的或然率可保守地估计为0.188.4=6×10-7。 Because distinguish average 8.4 DNA fragments are mixed probe drawing, so all of these fragments in one individual are present in a second randomly selected individual can be conservatively estimated the probability of 0.188.4 = 6 × 10- 7. 为此,这些图形提供了一个高水平的个体特异性-虽然並不象使用多位点探针或继后的与位点特异性探针杂交所能达到的那么高(参见上文)。 For this reason, these graphics provide a high level of individual-specific - though not, as the use of multi-locus probes or Following with site-specific probe hybridization can reach as high (see above). 合并的探针和个别位点特异性探针在研究细胞嵌合性,特别是对提供骨髓移植后追踪记忆留迹(engraphment)的供体对受体标志是很有用的;DNA混合实验(图12)表明,使用这些探针可检验出低水平的嵌合性。 Merger probes and individual locus-specific probes in the study of cell chimerism, especially after bone marrow transplant to stay track memory trace (engraphment) donor receptor flag is useful; DNA mixing experiments (Fig. 12) showed that the use of low levels of these probes can be tested chimeric.

下列实施例中所用的温度均为摄氏度(℃),且如英国专利公告2,166,445号中所述的探针33.6和33.15分别有下列的相符重复序列:(A)AGGGCTGGAGG,和AGAGGTGGGCAGGTGG Used in the following examples temperatures are in degrees Celsius (℃), and as British Patent Publication No. 2,166,445 the probes 33.6 and 33.15, respectively, have the following consensus repeat sequence: (A) AGGGCTGGAGG, and AGAGGTGGGCAGGTGG

实施例1该实施例叙述了DNA指纹图中特定之小随体片段的分离及性质一般方法按Jeffreys AJ等人〔Nature316,76-79(1985)〕所述的方法由白血细胞中分离DNA,並按Jeffreys等人(Am.J.Hum.Genet.39,11-24)所述的方法由Epstein-Barr病毒转染的、得自HPFH谱系之个体Ⅲ9的类淋巴母细胞系〔见Nilsson,K等,Int.J.Cancer8,443-450(1971)〕中分离DNA。 Example 1 This example describes the DNA fingerprints of a specific small and isolated nature of the fragment with the general method according to Jeffreys AJ et al [Nature316,76-79 (1985)] by the method according to the separation of white blood cells in DNA, press Jeffreys et al (Am.J.Hum.Genet.39,11-24) method described by Epstein-Barr virus transfected obtained from the individual pedigree HPFH Ⅲ9 of lymphoblastoid cell lines [see Nilsson, K, etc., Int.J.Cancer8,443-450 (1971)] isolated DNA. 按上述Jeffreys AJ等人的文献中所述方法完成限制性酶切和Southern印迹杂交。 According to the method described above Jeffreys AJ, et al literature complete restriction and Southern blot hybridization. 按Dretzen,G等人(Anal.Biochem,112,295-298(1981))所述的方法在DE81滤纸上电泳以分离双股DNA探针片段,並按Feinberg,AP等人(Anal.Biochem.137,266-267,(1984))所述的随机寡核苷酸预引发方法用32P标记之;再依照Jeffreys等人(Nature 314,67-73(1985))所述方法制备单股小随体探针33.15。 By Dretzen, G et al., (Anal.Biochem, 112,295-298 (1981)) according to the method in DE81 filter paper electrophoresis to separate the double-stranded DNA probe fragments, press Feinberg, AP et al., (Anal.Biochem. 137,266-267, (1984), random oligonucleotide) of the pre-initiation method using 32P-labeled; re in accordance with the Jeffreys et al (Nature 314,67-73 (1985)) the method for preparing single-stranded minisatellite 33.15 probe body.

克隆高可变片段g通过0.5%琼脂糖凝胶电泳分离用Sau 3A酶切的600μg个体Ⅲ9类淋巴母细胞系DNA,並按Yang RCA等人(Meth.Enzymol.,68,176-182,(1979))所述的方法在透析膜上经电洗脱收集有相应大小的部分。 Cloning hypervariable fragment g by 0.5% agarose gel electrophoresis with Sau 3A digested 600μg individual Ⅲ9 lymphoblastoid cell line DNA, press Yang RCA et al (Meth.Enzymol., 68,176-182, ( 1979)) according to the method by electroelution in dialysis membranes have a correspondingly sized portion collected. 经过两次制备性凝胶电泳后,片段g被纯化1000倍(得率为150ng DNA)。 After two preparative gel electrophoresis fragment g was 1000 fold purified (yield 150ng DNA). 将20ng如此部分纯化的DNA连接于60ng用BamHI酶切后分离的λL47.1臂(见Loenen WAM和Branmar,WJ,Gene 20,249-259(1980))上,于体外包装並接种在大肠杆菌WL95(803,supE、supF、hsdRk-、hsdMk+、tonA、trpR-、metβ,P2溶源菌株)上(见Loenen的上述文献及Jeffreys,AJ等,J.Mol.Biol.156,487-503(1982))以收集重组体。 The 20ng thus partially purified DNA was ligated to 60ng digested with BamHI after separation λL47.1 arms (see Loenen WAM and Branmar, WJ, Gene 20,249-259 (1980)), the in vitro packaged and plated in E. coli WL95 (803, supE, supF, hsdRk-, hsdMk +, tonA, trpR-, metβ, P2 lysogenic strain) on (see above Loenen literature and Jeffreys, AJ, etc., J.Mol.Biol.156,487-503 ( 1982)) to collect the recombinant. 通过与小随体探针33.15的噬斑杂交方法筛选所得的1500个重组噬菌体库。 By minisatellite probe plaque hybridization screening the resulting library of 1500 recombinant phage 33.15. 在大肠杆菌ED8910(803、supE、supF、recB21、recC22、hsdS)〔见Loenen的上述文献〕上再接种並筛选4个阳性噬菌斑,之后用Blattner等人(Science194,161-169(1977))的方法制备重组噬菌体DNA。 In E. coli ED8910 (803, supE, supF, recB21, recC22, hsdS) [see Loenen the above document] replated and screened on four positive plaques, followed by Blattner et al. (Science194,161-169 (1977) ) The method of preparing recombinant phage DNA. 将重组噬菌体g3的Sau3A插入片段再克隆到pUC13的BamHI位点〔Vieira J.和Messing J.,Gene19,259-268(1982)〕中,並使之在大肠杆菌JM83,Δ(recA-srlR)306::Tn10(见Matfield,M.(1983)Ph.D.thesis,University of Leicester〕的recA衍生株中增殖。 The Sau3A insert of recombinant phage g3 fragment was cloned into the BamHI site of pUC13 [Vieira J. and Messing J., Gene19,259-268 (1982)] and allowed to E. coli JM83, Δ (recA-srlR) 306 :: Tn10 (see Matfield, M. (1983) Ph.D.thesis, University of Leicester] recA derivative strain of proliferating.

DNA序列分析超声处理、按大小收集pλg3DNA並散弹枪式克隆到M13mp19〔见Yanis-Perron C.等Gene33,103-119(1985)〕的SmaⅠ位点中。 DNA sequence analysis was sonicated, size-collecting pλg3DNA and shotgun cloned into M13mp19 [see Yanis-Perron C., etc. Gene33,103-119 (1985)] SmaⅠ sites in. 为确定pλg3之随机重复区域的DNA序列,用双脱氧核苷酸链终止方法〔见Sanger F.等人,Proc.Nat.Acad.Sci.USA74,5463-5467(1977)和Biggin MD等人Proc.Natl.Acad.Sci.USA80,3963-3965(1983)〕分析12个随机克隆的序列。 To determine the random pλg3 of repetitive DNA sequence of the region, by the dideoxynucleotide chain termination method [see Sanger F. et al., Proc.Nat.Acad.Sci.USA74,5463-5467 (1977) and Biggin MD et al., Proc .Natl.Acad.Sci.USA80,3963-3965 (1983)] Sequence analysis of 12 random clones. 这些克隆中有8个是由pλg3的随机重复之小随体区域得来的,並用于限定相符重复序列。 There are eight of these clones by random repetition of small pλg3 come with the body region, and for defining consistent repeats. 通过与32P标记的侧接探针a和B杂交(见下面图1所述)检测含5′和3′侧翼区的M13克隆,並作序列分析以确认侧翼区域序列以及小随体的开始和终止点。 Flanked by hybridization probe a 32P-labeled A and B (see Figure 1 below) detect M13 clone containing the 5 'and 3' flanking region, and for sequence analysis to confirm the start sequence and flanking regions and minisatellite termination point.

结果自DNA指纹图中分离特定小随体用小随体探针33.15检测受HPFH(胎儿血红蛋白的遗传稳定性)影响之Jeffreys等人〔Am.J.Hum.Genet.,39,11~24(1986)〕所述Gujarati家系的个体Ⅲ9及她的DNA指纹图,其含有一8.2Kb多态性片段(片段“g”;图1A),该片段倾向于与前述文献〔Am.J.Hum.Genet.39,11-24(1986)〕所述的家系之其它成员共分离。 Since the results of DNA fingerprinting isolate specific small figure with the body by minisatellite probe 33.15 detected by Jeffreys HPFH (fetal hemoglobin genetic stability) influence of others [Am.J.Hum.Genet., 39,11 ~ 24 ( 1986)] Individual Ⅲ9 the Gujarati pedigree and her DNA fingerprint, containing a 8.2Kb polymorphic fragment (fragment "g"; Fig. 1A), which tends to fragment the aforementioned literature [Am.J.Hum. Genet.39,11-24 (1986)] Other members of the family of the total separation. 经过两次制备凝胶电泳由其它小随体片段中纯化出这一DNA片段。 After two preparative gel electrophoresis, the DNA fragment was purified from the other minisatellite fragments.

图1图解显示了通过凝胶电泳进行纯化的电泳图谱。 Figure 1 shows schematically purified by gel electrophoresis electropherogram. 用Sau3A酶切Jeffreys等人所述家系中个体Ⅲ9的DNA,並经制备凝胶电泳收集6-9Kb片段(部分1)。 Jeffreys et al was digested with Sau3A the pedigrees individual Ⅲ9 of DNA, and purified by preparative gel electrophoresis to collect 6-9Kb fragment (part 1). 再次电泳分离这些片段以得到部分2-5。 These fragments were separated by electrophoresis again to get 2-5. 用Sau3A酶切Ⅲ9DNA的各等分部分並将每一部分通过0.8%琼脂糖作电泳分离,之后与小随体探针33.15进行Southern印迹杂交。 Each aliquot was digested with Sau3A and Ⅲ9DNA of each part by a 0.8% agarose electrophoresis for separation after minisatellite probe 33.15 and Southern blot hybridization. 倾向于与HPFH共分离的片段g(8.2Kb)在部分3中被纯化了大约1000倍。 Tend to co-segregate with HPFH fragment g (8.2Kb) in section 3 was purified approximately 1000-fold.

将在Sau3A消化产物中检测出的、並按上述方法纯化得到的富含片段“g”约1000倍的部分克隆到上述Loenen及Brammar文献中所述的λL47.1中,之后在P2溶源性rec+大肠杆菌上增殖,以选择重组噬菌体。 The digested product was detected in Sau3A, purified in accordance with the method described above to give fragment rich "g" of about 1000 times the part cloned into Loenen and Brammar λL47.1 described in the literature above, after the P2 lysogenic E. coli rec + proliferation, to select recombinant phage. 经与探针33.15杂交以筛选所得克隆库。 33.15 After hybridization probe to screen the resulting clones library. 得到四个很小(直径小于0.1mm)的阳性噬菌斑,但能够以低效率(0-8pfu/噬菌斑)重新接种于recB、recC大肠杆菌上,以产生正常大小的噬菌斑(直径1mm)。 To give four small (diameter less than 0.1mm) of the positive plaques, but can be inefficient (0-8pfu / plaques) to re-inoculated recB, recC E. coli on to produce normal-sized plaques ( diameter 1mm). 进一步定性两个克隆(即λg1和λg3),並显示分别含有7.7和7.8Kb的Sau3A插入片段。 Two clones further characterized (i.e. λg1 and λg3), and displays each containing 7.7 and 7.8Kb of Sau3A insert. 这些克隆都是从g泳带得来的(见下述),但分别比g带短0.5和0.4kb。 These clones were obtained from g to swim with (see below), but with a shorter, respectively, than the 0.5 g and 0.4kb. 因为生长于recB、recC大肠杆菌上的λg1或λg3中没有Sau3A插入片段的大小不均一性,所以我们断定插入的部分已于它们在rec+大肠杆菌中最初的增殖期间由各克隆中丢失。 Since growth in recB, λg1 or λg3 recC E. coli on no Sau3A fragment insert size heterogeneity, so we conclude that the insertion portion has been lost from each clone during their initial proliferation in rec + E. coli.

用Blattner方法〔见Science194,161-169(1977)〕制备的λg1和λg3的得率很低(只有λL47.1重组DNA之正常得率的1%),此再次表明这类小随体克隆的异常生长性质。 By Blattner method [see Science194,161-169 (1977)] λg1 λg3 prepared and very low yield (only normal yield of recombinant DNA λL47.1 1%), this shows once again that this type of minisatellite clones abnormal growth properties. 故将Sau3A插入部分再克隆到pUC13〔见Vieira,J.和Messing J.,Gene19,259-268(1982)〕中並在大肠杆菌JM83的recA衍生株〔见Matfield M.,Ph.D thesis,University of Leicester(1983)〕中增殖,以使插入部分的重排降至最小。 Therefore, the Sau3A insert portion subcloned into pUC13 [see Vieira, J., And Messing J., Gene19,259-268 (1982)] and strains derived [see Matfield M., Ph.D thesis in E.coli JM83 of recA, University of Leicester (1983)] in the proliferation, so that the insertion portion to minimize rearrangement. 所得亚克隆即pλg3(见图1B),含有-7.1KbSau3A插入部分,其比g3中的插入部分短0.7Kb。 The resulting subclones i.e. pλg3 (see Fig. 1B), comprising -7.1KbSau3A insertion portion, than the part of the short 0.7Kb insert in g3.

小随体片段g的组织通过限制性酶切图(图1B)和DNA序列分析(图2)测定pλg3中小随体片段的结构。 Minisatellite fragment g tissue (Fig. 1B) and DNA sequence analysis by the restriction map (Fig. 2) Determination of small structures pλg3 minisatellite fragments.

图1B中显示了DNA片段g的组织。 Figure 1B shows the organization of DNA fragment g. 用限制性内切酶AluⅠ(A)、DdeⅠ(D)、HaeⅢ(H)、MboⅡ(M)、PstⅠ(P)和Sau3A(S)酶切绘制pλg3中Sau3A插入部分的基因图。 Endonuclease AluⅠ (A) with the restriction, DdeⅠ (D), HaeⅢ (H), MboⅡ (M), PstⅠ (P) and Sau3A (S) gene digested plotted in FIG pλg3 Sau3A insert portion. 其中没有HinfⅠ或RsaⅠ的切点。 None HinfⅠ or RsaⅠ cut points. 7.14Kb插入部分含有37bp序列(见图2)的171次串联重复外加747Kb的侧接DNA。 7.14Kb insertion portion contains 37bp sequence (see FIG. 2) of 171 tandem repeats plus side 747Kb grounding DNA. 5′侧翼区域含有倒转之Alu“组件”(画影线显示)的开始部分。 5 'flanking region containing the reverse of the Alu "Components" (hatched display) beginning section. 图中显示了侧翼探针a和b之唯一序列的原点。 The figure shows the origin of the flanking probes a and b of a unique sequence.

图2显示了高可变片段g的DNA序列。 Figure 2 shows the hypervariable fragment g of DNA sequences. 图中将5′和3′侧翼区及小随体之起始和终止部分的序列连同小随体之三个随机区域的重复序列一起显示出来。 In the Figure 5 'and 3' flanking regions and minisatellite sequence of the beginning and end portions of small repeat sequences, together with three random areas of the body displayed together. 倒位的Alu“组件”之开始部分以下面画线的大写字母显示,而5′侧翼区域中的几个简单序列区则未在下面画线。 Inversions Alu "Components" in capital letters at the beginning of the following line drawing show, while the 5 'flanking region of a few simple sequence region is not in the line drawing below. 显示了37bp小随体重复单元的相符序列(Con),並给出了各个重复单元与这一相符的差异。 Shows a consistent sequence 37bp minisatellite repeat units (Con), and gives consistent with differences in the respective repeating units. 区域C中的第二个重复单元含有一HaeⅢ切点(下面未画线的),故该区域跨越小随体中的固有HaeⅢ位点(图1B)。 Region C of the second repeating unit containing an HaeⅢ tangent point (not shown below the line), so that the region spanning minisatellite inherent HaeⅢ site (Figure 1B). 还以英国专利公开2,166,445号中所述相似方式显示相符重复序列与“核心”序列连成一线。 Also in British Patent No. 2,166,445 discloses a similar manner was consistent with repeated sequence of "core" sequence in a line.

克隆pλg3含有一个除有单一的固有HaeⅢ位点外缺乏其它限制性切点的6.3Kb小随体。 Pλg3 clone containing a single addition to outside sites HaeⅢ inherent lack of other restriction sites of 6.3Kb minisatellite. 该小随体是由37bp单元的171次重复序列构成的,此37bp单元包含序列GTGGGCAGG;这个序列恰好符合于先前鉴定为几个不同的人类小随体所共享之11-16bp核心序列(见英国专利公开2,166,445号及本文图2)的最无变化部分。 The minisatellite is 171 times by repeating units constituting the sequence 37bp, 37bp this unit comprising the sequence GTGGGCAGG; this sequence exactly conforming to 11-16bp core sequence previously identified for several different human shared with the body of the small (see British Patent Publication No. 2,166,445 and Fig 2 herein) of the unchanged portions. 重复序列不是完全同源的;对来自小随体内之几个随机选择的区域的序列分析表明其中有有限量的重复序列变异。 Repeat the sequence is not completely homologous; small randomly selected from the regional body with a few of the sequence analysis showed that there is a limited amount of repetition which sequence variations. 最多的变异包括可产生一33bp重复单元之再集合的4bp缺失,这种变异散布于一个以上的重复单元中。 Maximum variation comprises generating a 33bp 4bp deletion repeat unit of the collection, this variation spread in more than one of the repeating units. 一个变异片段(区域C中的A→C颠换)产生出唯一的固有HaeⅢ位点並因此可能仅见于一个重复单元中。 A variant fragment (region C A → C transversion) produce unique inherent HaeⅢ sites and therefore may only be found in one repeat unit. 小随体的开始和终未重复单元比固有重复显然更能与该序列歧异。 Start and end minisatellite repeat unit is not clearly better than the intrinsic repeat the sequence divergence.

pλg3中的小随体侧接有含正常密度之限制性切点的非重复DNA(图1B)。 pλg3 small side connected with the normal density-containing restriction sites for non-repetitive DNA (Figure 1B). 5′侧翼区的开始部分是由倒转的Alu组件之头组成的。 The beginning of the 5 'flanking region is composed of the head of an inverted Alu assembly thereof. 由杂交探针a和b限定的其余之5′和3′侧翼区(图1B)是独特的序列DNA並只杂交到总DNA(未显示数据)中的这一位点上。 By hybridization probes a and b define the remainder of the 5 'and 3' flanking region (Fig. 1B) is a unique sequence DNA and hybridize only to the total DNA (data not shown) in which a point on. 5′侧翼区则含有大量的简单序列DNA〔多嘌呤和(ACC)n〕(图2)。 5 'flanking region of the DNA sequence contains a large number of simple [more purine and (ACC) n] (Figure 2).

小随体片段g检测单一的多态性位点为检验是否完整克隆的小随体片段能用作杂交探针以特异地确定人DNA中的相应位点,可于人DNA竞争物存在下,将pλg3的Sau3A插入片段与用HinfⅠ(DNA指纹图法常规使用的限制性内切酶)酶切的人DNA杂交(图3)。 Minisatellite fragment g detects a single polymorphic sites to test whether a full clone of minisatellite fragments can be used as a hybridization probe to specifically determine the human DNA corresponding sites can compete under the presence of human DNA, The (conventional DNA restriction endonuclease fingerprinting method used) pλg3 the Sau3A insert with human DNA digested with HinfⅠ hybridization (Figure 3).

图3显示了用pλg3检测之多态DNA片段的孟德尔遗传特征。 Figure 3 shows the state with as many DNA fragments detected pλg3 Mendelian genetic characteristics. 用HinfⅠ酶切8μg人DNA的样品並通过0.8%琼脂糖凝胶电泳分离之。 Digested with HinfⅠ 8μg human DNA sample through a 0.8% agarose gel electrophoresis of. 在6%聚乙二醇和50μg/ml碱剪切的人胎盘DNA竞争物存在下,于65℃在1×SSC中将酶解产物与pλg3的Sau3A插入片段作Southern印迹杂交,並于杂交后于65℃在0.2×SSC中洗涤。 In 6% polyethylene glycol and 50μg / ml sheared human placental DNA base competitor exist, at 65 ℃ in 1 × SSC in the hydrolyzate with the Sau3A insert pλg3 Southern blot hybridization, and hybridization after 65 ℃ washed in 0.2 × SSC in. pλg3在所显示的所有个体中均检出一个杂合的单一位点。 pλg3 displayed in all individuals were detected in a heterozygous single site. 等位基因(由字母标示之)的遗传特征是孟德尔式的。 Alleles (indicated by the letter) is Mendelian genetic characteristics of.

在高强度洗涤条件下(0.2×SSC,65°),在所有被检的个体中检测出一个或两个杂交的片段。 In the high strength wash conditions (0.2 × SSC, 65 °), the detected one or two hybridizing fragments in all individuals subject. pλg3检测出了先前试验的Ⅲ9的亲属中的8.2Kb片段,进一步证实泳带g已被克隆(资料未显示)。 pλg3 detect the relatives of the 8.2Kb fragment of previous trials Ⅲ9 further confirmed by swimming with g has been cloned (data not shown). 在较低强度洗涤条件下(1×SSC,65℃),则检测出其它不太清楚地杂交的多态性DNA片段。 In the (1 × SSC, 65 ℃) lower strength wash conditions, the other polymorphic DNA fragments detected clearly not hybridize.

在高强度洗涤条件下由pλg3检测到的DNA片段是作为孟德尔遗传方式之单一位点的等位基因被分离的。 Washing conditions at high intensity detected by pλg3 to DNA fragments as a single Mendelian inheritance pattern of a point separated alleles. 这个位点不是性联的,而且没有很明显地显示出来,但在所研究的两个大家系中表现有不完全的性联(被试验的61个子代,在 This site is not sex-linked, and there is no clearly displayed, but the performance of the two lines we studied had incomplete sex-linked (61 progeny being tested in

=0.43时 = 0.43

=0.18;资料未示出);因此它表现为一个常染色体位点,並已定位在染色体7上(见表1)。 = 0.18; data not shown); it therefore behaves as a autosomal locus, and has been localized to chromosome 7 (see Table 1).

该小随体位点处的极端多态性变异用得自pλg3的插入片段筛选取自79个随机选择之英国白种人DNA的HinfⅠ酶解产物,通过一长35Cm的、浓度为0.7%的琼脂糖,首先对单一的、之后对14个人的储集样品(每人1μgDNA)进行电泳分析,以得到最大程度的等位基因分辨能力。 The small position with extreme polymorphic variation at the point of use pλg3 inserts from 79 randomly selected from the screening of the British Caucasians HinfⅠ hydrolysates of DNA through a long 35Cm, the concentration of 0.7% agar sugar, first single, after the reservoir 14 individual samples (per 1μgDNA) electrophoretic analysis, to obtain the greatest degree of allele resolution. 图4中显示了由这个群体样品中检出的不同等位基因的长度,连同每个等位基因中重复单元的估计数及等位基因频率。 Figure 4 shows the length of this group by the different alleles detected in the sample, together with the repeat units in each allele and the allele frequency estimate. 据此图4显示出小随体等位基因大小的分布情况。 Accordingly Figure 4 shows the distribution of minisatellite allele sizes. 最短的共有等位基因中小随体的长度是使用侧接的单拷贝探针a和b(图1),通过纯合子中这个片段的基因图测定的(数据未示出)。 Allele shared with the shortest length of the body is small using flanking single-copy probes a and b (FIG. 1), through the genetic map of this fragment in a homozygote assay (data not shown). 每个等位基因的重复数是近似的数目,且取决于小随体中37对33bp的比例。 The number of repetitions of each allele is approximate number, and depending on the ratio of minisatellites of 37 pairs of 33bp. 除去短的共有等位基因外,其余的等位基因被取样检验一次(42个等位基因)、二次(12个等位基因)、三次(12个等位基因)或四次(5个等位基因)。 Total removal of the short allele, the remaining alleles were sampled tested once (42 alleles), secondary (12 alleles), three (12 alleles) or four times (five allele). 如果所有这些等位基因都是同样罕见的,则这一分布与预测的(q=0.0081,2〔4d.f.〕=4.0)结果並没有显著差异,因此与存在于人群中的一个共有短等位基因(q=0.165)和103个同等罕见的等位基因(每个等位基因q=0.0081)的简单模式是一致的。 If all of these alleles are equally rare, the results did not predict the distribution of (q = 0.0081,2 [4d.f.] = 4.0) significant differences exist in the population and therefore a total short allele (q = 0.165) and 103 equally rare alleles (one for each allele q = 0.0081) is consistent with a simple model.

在这个人群样品中,可分辨出至少77个不同的等位基因,同时每个等位基因的重复数为14至525左右。 In this population sample, could distinguish between different alleles of at least 77, while the number of repetitions of each allele is about 14-525. 该人群样品中的纯合子都是最短等位基因的纯合子。 The population sample is homozygous homozygous for the shortest allele. 上述对等位基因数及罕见等位基因之平均频率的估计是受凝胶分辨能力限制的,该样品中不同长度等位基因的真实数目可能更大些。 Above estimates of the number of alleles and the average frequency of rare alleles are distinguished by gel capacity constraints, the real number of alleles of different lengths may be greater in the sample.

等位基因长度的分布似乎並不是完全随机的,但有可分为三类的证据(图4),即分为短(14-16次重复)、中(41-68次重复)和长(107-525次重复)等三类等位基因。 Allele length does not seem completely random, but there can be divided into three categories of evidence (Figure 4), which is divided into short (14-16 repeats), medium (41-68 repeats) and long ( 107-525 repetitions) and other three alleles. 对于定位于人胰岛素基因中5′端的高可变区来说,先前已在白种人中记录有等位基因长度的双峰分布〔见Bell,GI等人,Nature 295,31-35(1982)〕,但这种双峰分配在黑人中却不明显〔Lebo,RV等人,Proc.Nat.Acad.Sci.USA80,4804-4812(1983)〕。 For positioning in the human insulin gene 5 'end of the hypervariable region, the previously-recorded length alleles in Caucasians have a bimodal distribution of [see Bell, GI, et al., Nature 295,31-35 (1982 )], but this bimodal distribution in blacks is not obvious [Lebo, RV, et al., Proc.Nat.Acad.Sci.USA80,4804-4812 (1983)].

片段g的分离表明,DNA指纹图法中大的和高度多态性的DNA片段原则上能够适合于进行分子克隆,从而提供了适用于研究个别高可变区的位点特异性探针。 Fragment isolated g show the DNA fingerprinting method, large and highly polymorphic DNA fragments can be adapted to carry out the principles of molecular cloning, thus providing suitable for studying individual hypervariable locus specific probe. 虽然片段g可被克隆到噬菌体入中,但所得之克隆在rec+大肠杆菌上却显示了不正常生长性质。 Although fragments of g can be cloned into phage lambda, but it clone obtained from the rec + E. coli have shown the abnormal growth properties. 这将导致来自常规放大之人类基因库之小随体克隆的缺失。 This will lead to lack of conventional small amplification of the human gene pool from satellite clones. 已克隆的小随体当再克隆到recA大肠杆菌中的pUC13内时或于再克隆期间都是不稳定的,与片段g比较,最后的重组体pλg3已丢失了大约30%的单元。 During the cloned minisatellite when subcloned into pUC13 in recA E. coli or in recloned are unstable, compared with fragment g, the final recombinant pλg3 had lost about 30% of the units. 因此pλg3的物理图(图1)並不完全反映片段g的组织。 Physical map therefore pλg3 (Fig 1) does not entirely reflect fragment g tissue.

已克隆的DNA片段具有预期之小随体结构,证实片段g不是由较长随体DNA衍生的。 The cloned DNA fragment has the structure with small expectations, it was confirmed by the fragment g is not derived from a longer satellite DNA. 尽管在每个重复单元中均存在有“核心”序列。 Although there are "core" sequence in each repeating unit. 且在5′侧接区域中存在Alu序列的一部分,但在“竞争物”人DNA存在下,该已克隆的片段g可起到位点特异性探针的作用。 And a part of the Alu sequence present in the 5 'flanking region, but in the "competitor" presence of human DNA, the cloned fragment g could play the role of place-specific probe. pλg3不足以有效地检测其它含有核心的小随体,是由于在每个重复单元中存在有其它的非核心DNA,其可能干扰与其它小随体交叉杂交(见英国专利公告2166445号)。 pλg3 sufficient to effectively detect other core-containing minisatellites is due to the presence in each repeating unit other non-core DNA, which may interfere with cross-hybridization to other minisatellites (see British Patent Publication No. 2,166,445).

已克隆的小随体表现有极端的长度多态性,这可能是由于每个等位基因中重复单元之数目及37和33bp重复类型之比例这两方面的等位基因变异所至。 Cloned minisatellite surface prior extreme length polymorphism, which may be due to the number of repeat units in each allele and the ratio of 37 and 33bp repeat type of allelic variation both actions. 在158个染色体的随机人群样品中,可分辨出一个共有的和至少76个罕见的等位基因。 In the random population sample of 158 chromosomes, you can distinguish a total of at least 76 rare alleles. 这个位点要比迄今已定性的大多数或所有已克隆之人类高可变区都具有更大多态性(包括选择最初用于定义“核心”序列之相对短的已克隆小随体〔见美国专利公告2,166,445号〕)。 This locus has been characterized to date than most or all of the cloned human hypervariable region having a larger capital polymorphisms (including the selection for a relatively short initial definition of "core" sequences of cloned minisatellites [see U.S. Patent Publication No. 2,166,445]). 这一位点上的杂合性至少是96.6%,同时大多数杂合子是由短的共有等位基因产生的。 Heterozygosity at least on this one point is 96.6%, while the majority of heterozygous alleles by short total generated.

通过改变重复次数如通过DNA复制期间的滑动或由“核心”序列(一公认的重组信号)驱动的不等交换可产生这个位点上的新的等位基因。 By changing the number of repetitions during DNA replication, such as by sliding or by a "core" sequence (a recognized recombinant signal) driven by unequal exchange produces new alleles at this locus on. 在中性突变随机漂移假说下,参数4NeV(θ)(其中Ne是有效群体大小,V是每个配子突变为一新的长度等位基因的突变率)可使用无限等位基因模型(infinite allele model)〔见Ewens,WJ,Theor.Popul.Biol.3,87-112(1972)〕十分准确地由某群体样品中记录的不同等位基因数目估算出来。 In the neutral mutation random drift hypothesis, the parameter 4NeV (θ) (where Ne is the effective population size, V is a new mutation in each gamete length allele mutation rate) can be used infinite allele model (infinite allele model) [see Ewens, WJ, Theor.Popul.Biol.3,87-112 (1972)] the number of different alleles very accurately recorded by a group of samples estimated. 我们估算这个位点的θ是60-90(同时依据是否包括共有的短等位基因)。 We estimate this locus of θ is 60-90 (and according to whether to include common short allele). 因为人的Ne是~104V,故在这个位点上突变为新长度等位基因的突变率约为每个配子0.002。 Because human Ne is ~ 104V, so at this point mutant to the new length allele mutation rate of approximately 0.002 per gamete. 因为所检出的等位基因数目是受凝胶分辨能力限制的,而且没有一个无限数目的潜在的可分辨等位基因,所以上述的V值实际上是一个低估的数值。 Because the number of alleles detected is limited by gel resolution, and no potential of an infinite number of alleles can be distinguished, so the above-described V value is actually an underestimated value. 这个位点上小随体的平均长度是5Kb,因而每Kb小随体的突变(重组)率为>4×10-4,此与对其它较短的含核心小随体所估算的每Kb为10-4,以及人DNA的平均减数分裂重组率为每Kb约10-5差不多〔参见Jeffreys,AJ等,Nature314,67-73(1985)〕。 The average length of this locus minisatellite is 5Kb, therefore mutation per Kb minisatellite (recombinant) was> 4 × 10-4, this and other shorter core-containing body with a small estimated per Kb 10-4, and the average rate of meiotic recombination human DNA of about 10-5 per Kb almost [see Jeffreys, AJ, etc., Nature314,67-73 (1985)]. 可见,在这个小随体位点上新等位基因的产生率是非常高的,此与本发明人先前提出的这些富含核心区域可能是重组热点这一观点是一致的。 Visible on this point minisatellite alleles produce new rate is very high, and this with the present inventors have previously proposed these rich core areas may be recombination hotspots is consistent with this view. 短等位基因的突变率相对低些,此可解释为什么群体中最短等位基因漂移虽已达到一个显著的频率、但在此过程中却未受到不等交换的破坏。 Short allele mutation rate is relatively low, this may explain why the shortest allele population drift has exceeded a significant frequency, but in this process has not been damaged unequal exchange.

业已证明,DNA指纹图法对于个体鉴定及确证家族关系(如在父亲身份和移民争执中)是一种十分有效的方法。 Proven, DNA fingerprinting method for the identification and validation of individual family relationships (such as in paternity and immigration disputes in) is a very effective method. 该方法的准确度是由一条带被两个随机选择的个体共享的低平均概率X决定的。 The accuracy of this method is the low average probability of being shared by the two individuals randomly selected from a band X decision. 对于英国白种人,就大于4Kb之DNA指纹图HincⅠ片段来说;经验上估计X为0.2。 For British Caucasians, DNA fingerprinting is greater than 4Kb HincⅠ fragment of it; Project X 0.2 is experience. 当出现带在两个个体之间共有的情况下(即由于某条带有相似的迁移率和放射自显影强度,而导致一个个体的一条带与第二个个体中的带相匹配),便弄不清该共享的带是否代表某一小随体位点的相同等位基因。 As in the case with a total of between two individuals appear (i.e. due to a strip with a similar mobility and autoradiographic intensity, resulting in a single band of an individual with a second individual with matches), then figure out whether the shared bands represent identical alleles of a minisatellite points. 图4所示的等位基因分布使我们能够计算出这些>4Kb之等位基因的X;对于这个特定的小随体位点来说,X=0.016,其比由多位点DNA指纹图估计的值低一个数量级。 Allele distribution shown in Figure 4 allows us to calculate these> 4Kb of alleles X; for this particular point, minisatellite, X = 0.016, its ratio by multi-locus DNA fingerprinting estimated value one order of magnitude. 如果这一克隆的小随体是呈现于DNA指纹图中的典型位点上的,则在无关DNA指纹图间共享的大多数带即是由于不同小随体片段的偶然共迁移所致。 If this cloned minisatellite is in between unrelated DNA fingerprints that is shared by most of the band due to chance comigration of different minisatellite fragments in the DNA fingerprints due to render typical sites on. 这一重要的实践结果在于,在不能排除的案例-一例如在父亲身份争议中,就孩子中的所有父系带明确地存在于假定之父亲的DNA指纹图中的概率来说,据先前的计算其假性包涵的概率是很低的(X=0.2),而这又只是对真实概率(X=0.016)的一个粗略地过高估算。 The important practical results that can not be excluded in the case - for example, in a paternity dispute, to children of all paternal band clearly assumed the father's presence in the DNA fingerprints of probability, it is calculated according to the previous the probability is very low false indulgence (X = 0.2), which in turn is the true probability (X = 0.016) of a roughly estimate is too high.

使用DNA指纹图探针33.6和33.15,有可能记录到在大的人类血亲中有多达34个分散的常染色体位点。 Using DNA fingerprinting probes 33.6 and 33.15, it is possible to have up to 34 records dispersed autosomal loci in human blood in large. 对于大多数已检出的位点,在一组较大的(>4Kb)已分辨之DNA指纹图片段中,只有两个等位基因之一是可记录(显示)的,此提示小随体等位基因之间存在有很大的长短差异,同时许多等位基因是位于DNA指纹图的很难被分辨的(<4Kb)区域内。 For the majority of sites have been detected in a group of large segments of DNA fingerprinting picture (> 4Kb) has distinguished between, only one of the two alleles are records (show), and this prompted minisatellite There are great differences between the length alleles, while many alleles are located within the DNA fingerprint is difficult to be resolved (<4Kb) area. 这种情况亦见诸于已克隆的小随体;在这一位点上的HinfⅠ等位基因长度为1.7至20.4Kb,而且图4中所示的等位基因频率分布表明这个位点的等位基因在仅仅40%的个体之DNA指纹图中得以分辨,同时还有14%的人没有记录到显见的等位基因。 This situation is also found in the cloned minisatellite; at this point of a length of 1.7 to HinfⅠ allele 20.4Kb, and FIG allele frequency distribution in Fig 4 shows that the locus of the like alleles can be resolved in only 40% of individuals in the DNA fingerprints, as well as 14% of people do not record the apparent allele.

小随体探针33.6和33.15共检测到大约60个高可变位点,其中许多位点现已能适于克隆,以提供一个高度信息性的单一位点探针库,例如可用于人的连锁研究。 Minisatellite probes 33.6 and 33.15 were detected about 60 hypervariable loci, many of which can be adapted for cloning site has been to provide a highly informative single locus probes library, for example, for use in humans linkage studies. 使用完整的DNA指纹图亦可能对单个大家族中的遗传性疾病进行连锁分析。 Use complete DNA fingerprinting also possible linkage analysis of a single large family of hereditary diseases. 如果一个被检测的多态性片段表现与疾病共分离,即有必要克隆这个片段,以提供一个将连锁分析扩展到其它受影响之家族的位点特异性探针。 If one is detected polymorphic fragments were isolated and disease manifestations, namely the need to clone this fragment, in order to provide a linkage analysis will be extended to other affected family of site-specific probes.

在下面的实施例2中开始使用下述材料和方法。 In the following Examples 2 to start using the following materials and methods.

a)克隆一选择的大的小随体由40个随机选择的个体各制得5μg血细胞DNA並合并之,用Sau3A完全酶解並在0.6%琼脂糖凝胶中经制备电泳分离出5-15Kb片段,电洗脱后加于透析膜上透析。 large minisatellite a) selecting a clone from 40 randomly selected individuals 5μg of each blood cell DNA was prepared and pooled, digested to completion with Sau3A in a 0.6% agarose gel and isolated by preparative electrophoresis 5-15Kb fragments elute after power is applied to the dialysis membrane dialysis. 在第二个制备凝胶中电泳分离已纯化的部分,以除去所有痕量污染的小Sau3A片段。 In a second preparative gel electrophoresis separation of the purified components in order to remove all traces of the small Sau3A fragment contamination. 将50ng5-15Kb部分连接于100ng用BamHⅠ酶解后分离之λL47.1臂〔Loenen和Brammar,Gene20,249-259(1980)〕上,体外包装並构建成库,用人小随体探针33.6和33.15(见英国专利公告2,166,445号)筛选並按实施例1所述方法分离阳性噬菌斑,以得到MS系列重组噬菌体。 The λL47.1 arm 50ng5-15Kb part connected to 100ng of separation after hydrolysis with BamHⅠ [Loenen and Brammar, Gene20,249-259 (1980)], the in vitro packaging and build into a library, the employer minisatellite probes 33.6 and 33.15 (see British Patent Publication No. 2,166,445) in accordance with the method described in Example 1 Screening isolated positive plaques embodiment, in order to obtain MS series of recombinant phage.

b)分离小随体杂交探针用Sau3A酶解重组噬菌体DNA,並在低凝胶熔解温度琼脂糖(Sea Plaque)中经电泳由小载体片段中分离出大的插入片段。 b) Isolation of minisatellite hybridization probes Recombinant phage digested with Sau3A DNA, and the vector fragment was isolated by small in low melting temperature agarose gel (Sea Plaque) by electrophoresis in a large insert. 将含有插入物片段的凝胶条溶解于3倍体积水(65℃)中,並通过随机寡核苷酸引发法〔Feinberg和Vogelstein,Anal.Biochem.137,266-267(1984)〕用32P标记含10ng插入物片段之各等分部份。 Gel fragments containing the insert was dissolved in 3 volumes of water (65 ℃) in, and triggered by the random oligonucleotide method [Feinberg and Vogelstein, Anal.Biochem.137,266-267 (1984)] with 32P tag containing 10ng aliquot of each object is inserted fragments.

c)Southern印迹杂交按Jeffreys等人〔Am.J.Hum.Genet.39,11-24(1986)〕所述的方法限制性切断基因组DNA样品並进行电泳分离。 c) Southern blot hybridization by Jeffreys et al. [Am.J.Hum.Genet.39,11-24 (1986)] according to the method the genomic DNA sample is cut restriction and electrophoretic separation. 将DNA转移到Hybond-N(Amersham)上,用u/v照射以固定之並在0.5M磷酸钠、7%SDS、1mM EDTA(pH7.2)中于65°预杂交5分钟〔Church和Gilbert,Pro.Natl.Acad.Sci.USA81,1991-1995(1984)〕。 DNA was transferred to Hybond-N (Amersham), and with the u / v irradiation and to fix it in 0.5M sodium phosphate, 7% SDS, 1mM EDTA (pH7.2) at 65 ° prehybridized 5 minutes [Church and Gilbert , Pro.Natl.Acad.Sci.USA81,1991-1995 (1984)]. 于25μg/ml经剪切的单股人胎盘DNA竞争物存在下,将滤膜与0.5μg/ml32P标记的探针DNA(比活性为~109Cpm/ug DNA)杂交过夜。 At 25μg / ml sheared under a single strand of human placental DNA competitor exists, the probe DNA to the membrane and 0.5μg / ml32P labeled (specific activity of ~ 109Cpm / ug DNA) hybridized overnight. 杂交后,于65°在40mM磷酸钠、1%SDS(pH7.2)中洗滤膜,之后再于65°在0.1×SSC(15mM Nacl、15mM檬柠檬酸三钠,pH7.0)、0.01%SDS中以高强度洗涤。 After hybridization at 65 ° in 40mM sodium phosphate, 1% SDS (pH7.2) in the filter was washed, then again at 65 ° in 0.1 × SSC (15mM Nacl, 15mM citric trisodium citrate, pH7.0), 0.01 % SDS washing at high intensity. 于加强膜(intensifier screen)存在下,于-80°将滤膜放射自显影3小时至1周。 To strengthen the membrane (intensifier screen) in the presence of, at -80 ° Filters were autoradiographed 3-1 hours.

d)测定MS小随体的串联重复序列用超声波处理各MS重组DNA,选择大小为0.3-0.6Kb的片段並散弹枪式地克隆到M13mp19的SmaⅠ位点〔Yanis-Perron,Vieira和Messing,Gene33,103-119(1985)〕中。 d) tandem MS minisatellite repeats sonicated each MS recombinant DNA, selecting for fragments 0.3-0.6Kb size and shotgun cloned into the SmaⅠ site of M13mp19 [Yanis-Perron, Vieira and Messing, Gene33,103-119 (1985)] in. 与32P标记的MS插入物DNA作噬斑杂交以筛选重组的噬菌体。 And MS 32P-labeled insert DNA as a plaque hybridization to screen recombinant phage. 通过C-试验〔Winter和Fields,Nucleic Acids Res.8,1965-1974(1980)〕成对地比较自给定的MS重组体分离的10个强阳性单链噬菌体DNA;多数DNA落入两个互补的C-试验组内,因此其很可能是由λMS插入片段之长的、随机重复的区域来的。 By C- test [Winter and Fields, Nucleic Acids Res.8,1965-1974 (1980)] from a given pairwise comparison of MS recombinants isolated 10 strong positive single-stranded phage DNA; the majority fall into two complementary DNA C- within the test group, and therefore it is likely to be inserted by the λMS long fragment of, randomized to the repeated region. 用双脱氧核苷酸链终止方法〔Sanger,Nicklen and Coulson,(1977)Proc.Natl.Acad.Sci.USA74,5463-5467;Biggin,Gibson and,Hong,(1983)Proc,Natl.Acad.Sci.USA80,3963-3965〕测定得自两个互补组的各两个M13重组体的序列,以确定两条链上每个MS重组体的相符重复序列。 By the dideoxynucleotide chain termination method [Sanger, Nicklen and Coulson, (1977) Proc.Natl.Acad.Sci.USA74,5463-5467; Biggin, Gibson and, Hong, (1983) Proc, Natl.Acad.Sci .USA80,3963-3965] Determination of the sequence obtained from M13 recombinants each two groups of two complementary, in order to determine the consensus repeat sequence of each MS recombinant on both strands.

实施例2a)分离-选择的位点特异性高可变DNA探针实施例1中已述及了通过制备凝胶电泳分离,之后再克隆到噬菌体中这一克隆选自人DNA指纹图谱之片段的实施方案。 Example 2a) separated - the selected site-specific hypervariable DNA probes in Example 1 was addressed by preparative gel electrophoretic separation, and then subcloned into the phage clones selected from a fragment of human DNA fingerprinting of The implementation of the plan. 为分离较宽范围的小随体,可将选自40个随机选择之个体DNA的大小为6-15Kb的Sau3A片段克隆到BamHⅠ置换载体λL47.1〔Leonen and Brammar,“A bacteriophage lamda Vector for Cloning large DNA fragments made With several restricticn enzymes”Gene20,249-259(1980)〕中。 For the separation of a wider range of minisatellites, the size can be selected from 40 randomly-selected individuals Sau3A DNA fragment was cloned into BamHⅠ 6-15Kb replacement vector λL47.1 [Leonen and Brammar, "A bacteriophage lamda Vector for Cloning large DNA fragments made With several restricticn enzymes "Gene20,249-259 (1980)] in. 因为人小随体在其全长中显示有显著地等位基因变异(如实施例1中所证明的),所以使用合并的人DNA克隆大的Sau3A片段,应能使可克隆之高可变位点(其具有大的Sau3A等位基因)的数目达到最大。 Since human minisatellites is displayed in its full length in significant allelic variation (as demonstrated in Example 1), so the use of pooled human DNA clone large Sau3A fragments should enable hypervariable be cloned site (which has a large Sau3A alleles) reaches the maximum number. 经与人小随体探针33.15杂交,由含2000个重组体的文库中分离出5个噬菌体並用探针33.6检测出另外5个噬菌体,以得到MS系列的重组体(见表1)。 After the human minisatellite probe 33.15 hybridization, isolated from library containing 2000 recombinants out of 5 and phages detected by probes 33.6 additional 5 phage to obtain recombinant MS series (see Table 1).

为确定是否每个λMS重组体都能用作高可变小随体的位点特异性探针,在有人DNA竞争物存在下,将每个重组体的Sau3A插入片段与3个随机选择之个体的HinfⅠ酶解产物杂交,或与λMS32的AluⅠ酶解产物〔其小随体串联重复单元含有HinfⅠ位点(图5)〕杂交。 To determine whether each λMS recombinants can be used as hypervariable minisatellite locus specific probe body, in the presence of competitor DNA was, the Sau3A insert of each recombinant, and three randomly-selected individuals The HinfⅠ hydrolysates hybridization, or with AluⅠ λMS32 hydrolysates of [its minisatellite tandem repeat units containing HinfⅠ sites (Fig. 5)] hybridization. 10个重组体中有8个杂交到各个被试者DNA中的一个或两个大的可变DNA片段上。 10 recombinants in 8 subjects each DNA hybridized to one or two large variable DNA fragments. 用探针33.6检测到的重组体中有四个(λMS8、40、42和47)杂交到可变DNA片段之同一图形上,因而展示它们是衍生自同一高可变位点(图5,表1)。 Detected by probe 33.6 to recombinants four (λMS8,40,42 and 47) hybridized to the same figure on variable DNA fragments, thus showing that they are derived from the same hypervariable locus (Fig. 5, Table 1).

图5显示筛选适用作位点特异性高可变DNA探针之λMS重组体的一个例子。 Figure 5 shows the filter suitable for use as a site-specific hypervariable λMS an example of recombinant DNA probes. 按本文所述的材料和方法用HinfⅠ酶切2μg得自三个无关个体的DNA样品,通过0.8%琼脂糖凝胶作电泳分离並与32P标记的得自pλg3(实施例1)、λMS8和λMS40的Sau3A片段作Southern印迹杂交。 Materials and methods described herein by using HinfⅠ digested 2μg obtained from three unrelated individuals DNA samples, separated by 0.8% agarose gel electrophoresis for separation and labeled with 32P from pλg3 (Example 1), λMS8 and λMS40 The Sau3A fragment as Southern blot hybridization. 杂交后在0.1×SSC中(65°)洗滤膜,並于加强膜存在下放射自显影1天(左泳道)或1周(右泳道)。 After hybridization in 0.1 × SSC (65 °) in the filter was washed, and in the presence of the reinforcement film autoradiography 1 day (left panel) or 1 week (right lane). 应注意到的是,λMS8和λMS40检测出同一个高可变位点,而且如延长放射自显影时间,则可检测出其它几个可变DNA片段。 It should be noted that, λMS8 and λMS40 detected with a hypervariable loci, such as prolonged autoradiography and time, can be detected in several other variable DNA fragments.

其余的重组体λMS1、31、32和43衍生自不同的位点,但不包括先前定性的pλg3位点(见实施例1和图5)。 The remaining recombinant λMS1,31,32 and 43 derived from different sites, but does not include the previously characterized pλg3 locus (see Example 1 and Figure 5). 虽然这些λMS重组体都没有产生多可变片段的DNA“指纹图谱”,但经延长放射自显影爆光时间可见大多数探针与其它多态DNA片段有微弱的交叉杂交(图5)。 Although these λMS recombinants have not produced more variable fragment of DNA "fingerprint", but after prolonged autoradiogram exposing time most visible probes with other polymorphic DNA fragments have weak cross-hybridization (Fig. 5).

由探针33.15检出的两个λMS重组体即λMS3和λMS30,无论是否存在人DNA竞争物,均未能检测到用HinfⅠ或Sau3A酶切之人DNA中的特异性位点。 33.15 detected by the probe two λMS recombinants that λMS3 and λMS30, regardless of whether there were DNA competitor, were not detected or Sau3A digested with HinfⅠ of human DNA at specific sites. 基于延长放射自显影时间,两个重组体均检测出可变DNA片段之同样不明显的图形(资料未示出)。 Based autoradiogram extended time, two recombinants were detected in the same DNA fragment variable obvious pattern (data not shown). 我们没有进一步研究这一缺乏位点特异性现象,也没有进一步定性这两个MS重组体。 We have no further study of this phenomenon is the lack of site-specific, and no further characterize these two MS recombinants.

因此,尽管以前有报导认为难于克隆大的且不稳定的人类小随体〔如参见Nicholls等,Nucleic Acids Res.13,7569-7578(1985);Wyman等,Proc.Natl.Acad.Sci.USA77,6754-6758(1985)〕,但我们则已能够证明,至少有些在DNA指纹图谱中检测出的最大的和极可变的DNA可适合于在噬菌体λ中克隆,条件是通过在recBC大肠杆菌宿主中增殖使插入片段得以稳定。 Thus, despite previous reports considered difficult to clone large and unstable human minisatellites [see Nicholls et e.g., Nucleic Acids Res.13,7569-7578 (1985); Wyman, etc., Proc.Natl.Acad.Sci.USA77 , 6754-6758 (1985)], but we are already able to demonstrate that at least some detected in DNA fingerprints and highly variable maximum may be adapted to clone the DNA in bacteriophage λ, the condition is set by the recBC E.coli host proliferation make inserts to stabilize.

b)检测已克隆之小随体的重复序列单元测定来自每个λMS重组体之DNA插入物的随机小片段的序列,以确定是否每个克隆的高可变位点均是由随机重复的小随体构成的(见材料和方法)。 sequence repeat unit b) detecting the cloned minisatellite DNA insert determined from each λMS recombinants of random small fragments, to determine whether each cloned hypervariable locus are duplicated by small random minisatellite (see Materials and Methods) constituted. 已显示所有的λMS克隆主要是由长度范围为,9bp(λMS1)至45bp(λMS43)的串联重复单元构成的(图6)。 All λMS clones has been shown primarily by the length of the range, 9bp (λMS1) to 45bp (λMS43) tandem repeating unit (FIG. 6).

图6显示了λMS系列小随体的相符重复序列单元。 Figure 6 shows the consensus repeat sequence units λMS series minisatellite. 测定每个已克隆小随体的随机小片段,以限定相符重复单元。 Determined for each cloned minisatellite random small fragments, and to define the consensus repeat unit. 每个相符重复单元中可变位置显示为:R,r=A或G;Y,y=C或T;N,n=任何碱基。 Each repeating unit matches the variable position is shown as: R, r = A or G; Y, y = C or T; N, n = any base. MS8中的小随体包含两个共同散布的、29和30bp长的重复单元。 The minisatellite in MS8 consists of two common interspersed repeat units 29 and 30bp length. 图2中还显示了pλg3的相符重复序列。 Figure 2 also shows the consensus repeat sequence of pλg3. 每个重复序列均与核心序列对齐,同时用大写字母显示配对者,还揭示了根据该核心与pλg3、λMS31和λMS32比较而检出的一新的核心序列。 Each repeat sequences were aligned with the core sequence, while the pair were displayed in capital letters, but also reveals a new core sequence based on the core and pλg3, λMS31 and λMS32 comparing detected.

小随体中的重复单元都不是完全同源的,此与在以前测定了序列的小随体中所见到的重复单元之间的变异情况是一致的(见英国专利公告2,166,445号和本文实施例1)。 Minisatellite repeating units are not entirely homologous, and in this variation is measured before repeating unit minisatellite sequences is seen between the same (see British Patent Publication 2,166,445 number and Example 1 herein). 特别值得注意的是,λMS8中的小随体是由两个紧密相关的、但有不同长度(29和30bp)的重复单元搀合排布构成的(图6)。 Of particular note is that, λMS8 the minisatellite is comprised of two closely related, but different length (29 and 30bp) mix together the repeating units constituting the arrangement (Fig. 6). λMS重组体中小随体的端点尚未确定。 Endpoint λMS recombinant small satellite has not been determined.

据此,已分离了5个新的高可变位点,每个位点都主要包含一串联重复的小随体。 Accordingly, have been isolated five new hypervariable loci, each locus are mainly contains a tandem repeat minisatellite. 正如己预期到的,pλg3、λMS1、λMS31和λMS32的串联重复单元每个都含有核心序列(图6)。 As has been expected to, pλg3, λMS1, λMS31 and λMS32 tandem repeating units each containing the core sequence (Fig. 6). 然而,核心序列的拷贝是不完善的,而且比较这些小随体的重复单元並没有明确地限定与这些大的和极端变异的位点优先关联的新核心序列。 However, copies of the core sequence are imperfect, and the repeating unit compares these minisatellites do not clearly define a new core sequence with these large and extreme variability of loci associated priority. 有可能是宽范围的核心衍生物与大的小随体相关联。 There may be a wide range of core derivatives with large minisatellites associated. 特别值得注意的是,由探针33.6〔其由核心序列之离散型式的串联重复组成(见英国专利公告2,166,445号)〕检测出的小随体λMS8和λMS43含有高度离散型式的核心序列。 Of particular note is that the probes 33.6 [in series composed of discrete repetitive pattern of the composition of the core sequence (see British Patent Publication No. 2,166,445)] core detected minisatellites λMS8 and λMS43 containing highly discrete type sequence. 另外,比较λMS8、λMS43和33.6並不能揭示优选与这三个位点相关联的新的特异性序列(资料未示出)。 In addition, the relatively λMS8, λMS43 and 33.6 does not reveal new specific sequence preferably three loci associated (data not shown). 很明显,详尽地探究与极端可变小随体(特别是用探针33.6检测出的小随体)相关联的其它序列特征,将需要分析更大量的克隆小随体图谱。 Obviously, an exhaustive inquiry and extreme variable minisatellites (especially with the probe detected 33.6 minisatellite) other sequence features associated with the need to analyze a lot more clones minisatellite maps.

c)孟德尔遗传特征及用克隆小随体检测之位点的变异性已分离的5个新的小随体λMS1、8、31、32和43各检测一个大的和可变的位点。 c) Mendelian genetic characteristics and cloning minisatellite loci detection of variability detached five new minisatellites λMS1,8,31,32 and 43 each detection of a large and variable sites. 通过分析由CEPH程序提供的大的水平谱系中的分离情况,已进一步证实了所有位点的孟德尔式遗传特征(图7)。 By analyzing a large horizontal CEPH pedigree provided by the separation of the program, has been further confirmed by Mendelian inheritance of all loci feature (Fig. 7).

图7显示由λMS32探测之高可变位点的孟德尔式遗传特征。 Figure 7 shows the Mendelian genetic characteristics hypervariable loci detection of the λMS32. 用AluⅠ酶解采自CEPH家族1413的1μg DNA样品並Southern印迹杂交到32P标记之λMS31的Sau3A插入片段上。 AluⅠ digested with 1μg DNA samples taken from CEPH family 1413 and Southern blot hybridized to λMS31 the 32P-labeled Sau3A inserts. 这个家族的DNA样品在该位点处是全部带有信息性的。 This family of DNA samples at this site is that with all of the information.

通过分析在随机选择之英国人之间共享的等位基因频率,已估计了适于每一探针之等位基因的变异性程度(图8)。 By analyzing shared between randomly-selected British allele frequencies of each allele has been adapted to estimate the variability in the extent of the probe (Fig. 8). 图8提供了对MS43探测之高可变位点处等位基因变异性的估计,用HinfⅠ酶解的采自20个随机选择之英国人的1μg DNA样品並与λMS43作Seuthern印迹杂交。 Figure 8 provides at hypervariable loci detection of allelic variability MS43 estimates HinfⅠ digested with 1μg DNA samples collected from 20 randomly-selected British and make Seuthern blot hybridization with λMS43. 所有个体都是杂合性的,此表明在这个高可变位点处杂合性水平是很高的(H>0.86,P>0.95)。 All individuals are heterozygous, this indicates that the level of heterozygosity at this hypervariable locus is at a high (H> 0.86, P> 0.95). 将每一个体中的等位基因与6个最接近的邻居的等位基因进行比较(基于放射自显影)来估算个体间共享之等位基因数,从而更精确地估计杂合性水平。 The alleles in each individual with the six nearest neighbor comparing alleles (based on autoradiogram) to estimate the number of shared alleles between individuals, so as to more accurately estimate the level of heterozygosity. 例如,个体5中的较大等位基因似乎存在于7中,但不存在于个体2,3,4,6和8中。 For example, the larger allele in individual 5 seems to be present in 7, but not in individuals 2,3,4,6 and 8. 相似地,个体5中的较小等位基因並不存在于2-4或6-8中。 Similarly, the smaller allele in individual 5 is not present in 2-4 or 6-8. 对于所有被检的个体,s=0.11,由此可估算出平均等位基因频率q=1-(1-s)1/2=0.056,且杂合性为(1-q)=94%。 For all of the subject individual, s = 0.11, whereby the mean allele frequency estimate q = 1- (1-s) 1/2 = 0.056, and the heterozygosity as (1-q) = 94%. 由于受凝胶电泳分辨能力的限制,这只是对杂合性的一个最保守的估算。 Due to the limitation of the resolving power by gel electrophoresis, which is heterozygous for one of the most conservative estimate.

所有位点都是高可变的,其杂合性范围为90%(λMS8)至99%(λMS31)(见表1)。 All loci are highly variable, which heterozygosity range 90% (λMS8) to 99% (λMS31) (see Table 1). 比较在15和150个个体中分辨出的等位基因谱,进一步分析了等位基因长度变异的程度(图9)。 Comparative distinguish at 15 and 150 individuals alleles spectrum, further analysis of the extent of allelic length variation (FIG. 9). 图9显示了对人DNA库中高可变位点处等位基因长度变异的分析结果。 Figure 9 shows a library of human DNA hypervariable allelic length variation at the site of analysis results. 用AluⅠ(用于λMS32)或HinfⅠ(用于所有其余的探针)酶解采自某单一个体(a)、15个人的样品池(b)和150个人的样品池(c)的10μg DNA样品並与所指出的位点特异性高可变探针作Southern印迹杂交。 With AluⅠ (for λMS32) or HinfⅠ (for all remaining probes) hydrolysis were collected from a single individual (a), 15 individuals of the sample cell (b) and 150 individual sample cell (c) of 10μg DNA samples and with the indicated locus-specific hypervariable probes for Southern blot hybridization. 所有个体都是随机选择的北欧人。 All individuals are randomly selected Nordic. 单一个体的样品包含于样品池b内,所有池b内的样品又都包含于池c内。 Single sample of individuals included in the sample cell b, b sample within all pools are both included in the pool of c. 应指出的是,λMS43探测到由标记为0之短HinfⅠ等位基因代表的第二可变区;尚未对这个区域作进一步定性。 It should be noted, λMS43 detected by labeled alleles of 0 representative of a second short HinfⅠ variable region; yet in this area for further characterization.

对于最小可变位点λMS8,在15个人的样品池中可分辨出2个长度为5.3和7.2Kb的优势等位基因,外加7个较低频率等位基因。 For a minimum of variable sites λMS8, 15 individual samples in the pool can distinguish two lengths of 5.3 and 7.2Kb the dominant allele, plus 7 lower frequency alleles. 两个优势等位基因的出现並不是由于在这15个人中的抽样误差造成的,这一点可通过它们在150个个体中具有相似的优势並且有较大范围的低频率等位基因存在得以证明。 There are two advantages of the low frequency allele alleles appear in these 15 individuals was not due to the sampling error caused by this may have similar advantages in 150 individuals and has been proved that a wide range of . 相似地,λMS43(94%杂合性)显示有7个被15和150个个体共享的较大等位基因,连同许多较小等位基因。 Similarly, λMS43 (94% heterozygosity) showed seven shared by 15 large and 150 individual alleles, along with many smaller allele. 相反,对于最可变的位点λMS1、31和32以及pλg3,其估算的杂合性为97-99%,未显示有以相等强度被两个个体池共享的、具显著群体频率的等位基因(上文所述的pλg3的最短1.6Kb等位基因除外),因此这些位点的等位基数目很可能是很大的。 In contrast, for the most variable loci λMS1,31 and 32 and pλg3, its estimated heterozygosity 97-99%, does not appear to have equal intensity pool shared by two individuals, a significant population frequency of allelic gene (described above except 1.6Kb shortest pλg3 allele), and therefore the number of these sites is likely allelic great. 例如,在150个个体的样品池中可分辨出至少50个不同的λMS32等位基因;由于凝胶电泳分辨力的限制,这只是对等位基因总数的一个最小估算。 For example, in the 150 individual sample cell can distinguish at least 50 different λMS32 alleles; due to the limitation of the resolution of gel electrophoresis, which is a minimum estimate of the total number of alleles.

白种人中,等位基因在这些高可变位点之间的长度分布也是不同的(见图9和表1)。 Caucasians, the allele length distribution between these hypervariable loci is different (see FIG. 9 and Table 1). 大多数λMS31的等位基因,在5.5-9.3Kb这一相对窄的长度范围内分布相当均匀,而λMS1的等位基因则变化很大,长度范围为2-20Kb。 Most λMS31 alleles within 5.5-9.3Kb this relatively narrow distribution of relatively uniform length, and the allele is λMS1 vary widely, a length in the range of 2-20Kb. 在有些情况下,有证据显示等位基因长度分布的不均一性;特别是λMS43显示有如上面实施例1中所述的5-6Kb和8-14Kb这两类占优势的等位基因长度范围,且pλg3显示有1.6-1.7、2.8-3.6和5-15Kb这样三类长度范围。 In some cases, there is evidence of heterogeneity of allele length distribution; in particular λMS43 display like 5-6Kb described in Example 1 and 8-14Kb these two types of dominant allele length of the above embodiment, and pλg3 showed 1.6-1.7,2.8-3.6 and 5-15Kb categories such length.

如上面所指出的,已探测到的位点是极端可变的,並确实是迄今从人DNA中分离之位点中最具多态性的。 As noted above, the loci detected are extremely variable and indeed are sites so far isolated from human DNA in the most polymorphic.

d)高可变位点的染色体分配高可变位点特异性探针没有一个杂交到啮齿动物DNA上,因此可在人一啮齿动物体细胞杂种中追踪到这些位点的分离(见图10和表2)。 d) Chromosome assignment of hypervariable loci hypervariable locus-specific probes not hybridized to a rodent DNA, and therefore can be traced to the separation of these sites (see Fig. 10 in a human rodent somatic cell hybrids and Table 2). 6个位点分配于4个不同的常染色体上,其中染色体1和7各携带两个高可变位点。 6 sites distribution on the four different autosomes, chromosomes 1 and 7 each of which carries two hypervariable loci.

图10显示了在用AluⅠ酶切之人一啮齿动物体细胞杂种DNA的泳道中,由λMS32探测出之高可变位点的分离情况。 Figure 10 shows the person with AluⅠ digested a rodent somatic cell hybrid DNA lanes separated by λMS32 detected cases of hypervariable loci. 这一位点与人染色体1共分离(见表2)。 This is a point of human chromosome 1 were isolated (Table 2). 引人注意的是,杂种细胞系MOG2C2携带有λMS32和λMS1两者的两个等位基因(资料未示出)。 Notably, hybrid cell lines carrying both MOG2C2 λMS32 and λMS1 two alleles (data not shown). 这一现象强有力地提示:两亲代的人染色体1的拷贝已被保留于杂种中,而且,因所有其它阳性杂种在每一位点上只含有单个等位基因,这就为λMS1和λMS32的同线性提供了有力的证据。 This phenomenon strongly Tip: both parental copies of human chromosome 1 have been retained in the hybrids, and, because all other positive hybrids at each point contains only a single allele, which for λMS1 and λMS32 of synteny provided strong evidence. 杂种F4SC13c112含有染色体1p但是λMS32却为阴性;因此认为λMS32暂时定位于染色体1q上。 Hybrid containing chromosome 1p but λMS32 F4SC13c112 was negative; therefore considered λMS32 temporarily to chromosome 1q.

图10中所用的编号分别是:1.人2 16.人12.仑鼠 17.大鼠3.小鼠 18.DUR4,3 Figure 10 numbers used are: 1 person 2 people 12. 16. 17. hamster mouse rat 3. 18.DUR4,3

4.FG10 19.FIR55.PCT BA18 20.C4A6.1α9498 21.DUR4R37.SIF4A24EI 22.3W4c158.SIF15P5 23.克隆219.CTP34B4 24.WILF110.TWIN19D12 25.F4SC13c111.TWIN19F9 26.SIF4A3112.TWIN19F6 27.FST9/1013.TWIN19C5 28.HORL411B614.MOG2C2 29.HORP9.515.MOG2E5表2显示人一啮齿动物体细胞杂种中高可变位点的染色体分配情况。 4.FG10 19.FIR55.PCT BA18 20.C4A6.1α9498 21.DUR4R37.SIF4A24EI 22.3W4c158.SIF15P5 23. cloning 219.CTP34B4 24.WILF110.TWIN19D12 25.F4SC13c111.TWIN19F9 26.SIF4A3112.TWIN19F6 27.FST9 / 1013. TWIN19C5 28.HORL411B614.MOG2C2 29.HORP9.515.MOG2E5 Table 2 shows the distribution of human chromosome a rodent somatic cell hybrids hypervariable loci.

表2中所列体细胞杂种序号分别是:1,DUR4.3;2,FIR5;3,C4A;4,DUR4R3;5,3W4c15;6,克隆21;7,WILF.1;8,F4SC13c112;9,SIF4A31;10,FST9/10;11,HORL411B6;12,HORP9.5;13,FG10;14,PCTBA18;15,1α9498;16,SIF4A24E1;17,SIF15P5;18,CTP34B4;19,TWIN19D12;20,TWIN19F9;21,TWIN19F6;22,TWIN19C5;23,MOG2C2;24,MOG2E5且表中所使用之符号的意义是:+,存在有染色体或高可变位点;-,没有染色体或位点;M,只于某些细胞中存在染色体或用Southern印迹杂交法只能微弱地检测出的位点;N,未检测染色体或结果不肯定,未检测位点;P,存在短臂;D,因7/X转位而存在染色体7Pter→q22。 Listed in Table 2, the body cell hybrids numbers are: 1, DUR4.3; 2, FIR5; 3, C4A; 4, DUR4R3; 5,3W4c15; 6, clone 21; 7, WILF.1; 8, F4SC13c112; 9 , SIF4A31; 10, FST9 / 10; 11, HORL411B6; 12, HORP9.5; 13, FG10; 14, PCTBA18; 15,1α9498; 16, SIF4A24E1; 17, SIF15P5; 18, CTP34B4; 19, TWIN19D12; 20, TWIN19F9 ; 21, TWIN19F6; 22, TWIN19C5; 23, MOG2C2; 24, meaning MOG2E5 and the symbols used in the table are: +, presence of chromosome or hypervariable locus; -, no chromosome or locus; M, only or chromosomal locus by Southern blot hybridization only weakly detected in some cells; N, undetected chromosomal or results are not sure, no detection sites; P, the existence of the short arm; D, because 7 / X The presence of the chromosomal translocation 7Pter → q22. 给出了每个位点特异性高可变探针之推断的染色体分配,连同明确地提供信息的细胞杂种部分,这些杂种对于存在或不存在完整的染色体或高可变位点来说都是一致的。 Gives each locus-specific hypervariable probes deduced chromosome assignment of, along with clear information of cell hybrids portion, these hybrids for the presence or absence of a complete chromosome or hypervariable locus for both consistent. 对于每个高可变探针,用这一分析法除一个外所有染色体均被多重排除。 For each hypervariable probe, using the analysis of all chromosomes except one are multiple excluded.

λMS1杂交到含染色体1p的杂种8(F4SC13c112)上,提示λMS1是位于1p上。 λMS1 hybridized to chromosomes 1p hybrids containing 8 (F4SC13c112), the prompt λMS1 is located on 1p. 相反λMS32则没有杂交到杂种8上,提示其定位于1q上。 Instead λMS32 is not hybridized to hybrid 8, suggesting that it is positioned on 1q. pλg3没杂交到含染色体7pter→q22的杂种2(FIR5)上。 pλg3 not hybridized to 2 (FIR5) 7pter → q22 on chromosome containing hybrids. 再者,杂种JDA13.1和含7pter→q31.3的JDA3及7q31.3→qter对于pλg3分别是阴性或阳性的(资料未给出),pλg3被定位于7q31.3→qter。 Furthermore, the hybrid JDA13.1 containing 7pter → q31.3 and 7q31.3 → qter of JDA3 for pλg3 negative or positive, respectively (data not shown), pλg3 is located in 7q31.3 → qter. 反之,λMS31杂交到FIR5和JDA13.1上但未杂交到JDA3上,此提示,其定位于7pter→q22。 Conversely, λMS31 hybridizes to FIR5 and JDA13.1 but the hybridized to JDA3, this prompt, localized to 7pter → q22. 对大的CEPH血亲关系者中所作连锁分析进一步证实了这一缺乏束集(Clustering)的情况;分析表明,在各对同线性标志之间均没有明显的连锁关系(对于每对在>0.35时Z>-2,资料未示出)。 For large CEPH kinship were made in the case of linkage analysis further confirmed the lack of bundles (Clustering); the analysis showed that among the same linear marks are no obvious linkage (for each pair at> 0.35 Z> -2, data not shown).

所有6个克隆的小随体位点,包括实施例1中所述的pλg3,都是常染色体的和分散的;所发现之小随体的两同线性对中,均未显示明显地成对连锁。 All six cloned minisatellite points, including pλg3 described in Example 1, are autosomal and dispersed; two syntenic minisatellites found the pair, the pair of chain did not show significantly . 这种常染色体定位和束集缺乏与以前所作DNA指纹图谱的分离分析结果完全一致,其显示由多核心探针探测之大量高可变DNA片段均衍生自多数的、分散的常染体位点,但不能从DNA指纹图谱中推断出个别片段的染色体定位。 This lack of autosomal positioning and bundles separated by DNA fingerprinting analysis consistent with previous results, which is displayed by the large number of highly variable DNA fragment probing the multi-core, derived from the dispersion of autosomal majority position point, but can not be inferred from the chromosomal localization of individual fragments of DNA fingerprinting.

正如所预料的,如果迄今已定位的已克隆小随体随机分散于人基因组上,则很可能是得自于大的人常染色体(表1)。 As expected, if the date has been positioned cloned minisatellite randomly dispersed in the human genome, it is likely to be derived from the large human autosomes (Table 1). 在没有精确地区域性定位之情况下,这些小随体仍有可能是优先定位在着丝粒和端粒上,它们富集了有简单序列的卫星DNA。 In the absence of precise regional localization of these minisatellite priority may still be positioned in the centromeres and telomeres, which is enriched in simple sequence satellite DNA. 然而,对小鼠之重组的纯系细胞菌株中DNA指纹图谱片段所作分离分析表明,鼠的小随体並没有优先与着丝粒或端粒结合,而且很可能是它们的人DNA对应物也有相似地分散情况。 However, recombinant inbred strains of mice cells in DNA fingerprint fragments separated by analysis showed that mouse minisatellites not a priority with the centromere or telomere binding, and it may be their DNA counterparts who have similarly dispersed situation.

实施例3小随体探针的敏感性及法医学应用业经试验过的串联重复之小随体探针是非常灵敏的,此大概是因为杂交探针和靶小随体DNA之重复性质所致。 Example 3 minisatellite probe sensitivity and forensic applications already tried to repeat the series minisatellite probes is very sensitive, this is probably because of the repetitive nature of the hybridization probe and target DNA of minisatellite due. 杂交过夜后得到的Southern印迹放射自显影信号在探针DNA浓度>0.1ng/ml时达到饱和(资料未显示),而且能够很容易地从60ng或更少的人基因组DNA得到信号(见图11A)。 After hybridization overnight resulting Southern blot autoradiographic signal is saturated at probe DNA concentrations of> 0.1ng / ml (data not shown), and can easily obtain a signal (see FIG. 11A from 60ng or less of human genomic DNA ). 相似地,依据被试个体的基因型,这些探针能探测2%或更少的某一个体DNA与其它个体DNA的混合物(见图11B)。 Similarly, according to the genotype of the individual subjects, these probes can detect 2% or less of an individual's DNA with the DNA mixture of other individuals (see FIG. 11B).

这些位点特异性小随体探针的敏感性和变异性使之尤为适用于法医学。 These site-specific minisatellite probe sensitivity and variability make it particularly suitable for forensic science. 图11C显示出对取自两个遭强奸並被杀害之受害者身上的精液污斑所作分析,其中由第二个受害者身上回收的DNA如用常规的DNA指纹图谱分析法(其每次试验至少用0.5μg DNA)(见UK专利公告2,166,445号)检测是不够用的。 Figure 11C shows by analysis of semen stains from the victim had been raped and killed two of which recovered from the body of the second victim's DNA as with conventional DNA fingerprinting analysis (which each test At least with 0.5μg DNA) (see UK Patent Publication No. 2,166,445) detection is not enough.

为此,图11示出对位点特异性探针之敏感性的估价以及其在强奸/凶杀双重犯罪案例之法医学分析方面的应用。 To this end, Figure 11 shows the sensitivity of the position of the point of valuation specific probes as well as its double criminality in the case of rape / murder forensic analysis applications. 图11A中显示用降低量的、经HinfⅠ酶切的人DNA与32P标记的MS1之插入片段作Southern印迹杂交,于加强膜存在下滤膜放射自显影1周。 Figure 11A shown with a reduced amount of, by HinfⅠ digested human DNA insert MS1 with the 32P-labeled Southern blot hybridized, in the presence of strengthening membrane filter autoradiographed for 1 week. 图11B是以所指出的比例将降低量的个体A的DNA与4μg个体B的DNA混合,並依图11A所示与λMS1作Southern印迹杂交。 Figure 11B is indicated ratio will reduce the amount of individual A DNA mixed with 4μg DNA of individual B, and according to FIG. 11A and λMS1 Southern blot hybridization. 图11C中所示受害者X和Y是被强奸並继而被杀害的。 Figure 11C victims X and Y is shown being raped and then killed. 嫌疑犯Z被指控杀害了Y,並经法医学验证提示X和Y是被同一个人杀害的。 Z was accused of killing suspects Y, and by the forensic authentication prompt X and Y are the same person killed. 由下列法医学标品中分离DNA並进行分析:a,死后两天采取的头发;b,由X的阴毛上回收的精液与阴道液的混合物;c,由嫌疑犯Z身上采集的新鲜血液;d,Y死后一天采集的心脏内血液;e,用阴道拭子自Y阴道内采集的带有精液、血液和阴道材料的标本;f,从Y的裙子上采集的带有精液和血液的污斑材料,样品a和b已于4℃干燥储存了三年,样品d和e已于4℃干燥储存了两个月,且样品f也于4℃干燥储存了两个月。 Separated by the following standard products in forensic DNA and analyzed: a, taken two days after the death of hair; a mixture of b, recovered from the X pubic hair semen and vaginal fluid; c, Z fresh blood from suspects who collected; d within one day after the death of the heart blood collected Y; e, using vaginal swabs collected from the inside Y vagina with semen, blood and vaginal specimens of materials; f, collected from the Y's skirt dirty with semen and blood spot materials, samples a and b were 4 ℃ dry storage for three years, d and e samples were stored dry for two months 4 ℃, and the sample f also at 4 ℃ dry storage for two months. 依Gill、Jeffreys和Werrett的方法〔Nature318,577-579(1985)〕提取DNA,用HinfⅠ酶切,並与32P标记的λMS1插入片段作Southern印迹杂交。 By Gill, Jeffreys and Werrett method [Nature318,577-579 (1985)] to extract DNA, digested with HinfⅠ, with 32P-labeled λMS1 insert Southern blot hybridization. 除样品e(0.1μg DNA)和样品f(0.04μg DNA)外,其它各份样品均分析0.8μg DNA。 In addition to the samples e (0.1μg DNA) and sample f (0.04μg DNA), the respective parts of the other samples were analyzed 0.8μg DNA. 于加强膜存在下,放射自显影一周。 Strengthening presence in the film, autoradiography week. 注意到精液污斑b、e和f各含两个不属于受害者的杂交DNA片段。 Noting semen stains b, e and f, each containing two hybridized DNA fragments do not belong to the victim. 另外,样品b含有得自阴道DNA的受害者的等位基因。 In addition, sample b contains the victim's alleles derived from vaginal DNA. b、e和f中的精液等位基因是不能区分的,提示X和Y确实是被同一个人强奸的。 b, e and f in semen allele is indistinguishable, suggesting that X and Y are the same person indeed raped. 嫌疑犯Z的等位基因並不与精液污斑的等位基因相符合。 Suspect Z allele is not consistent with semen stains allele.

这些结果进一步经与λMS31杂交(资料未示出)並通过对大量法医材料的DNA指纹图谱分析得以证实。 These results further consultation with λMS31 hybridization (data not shown) and by a large number of forensic DNA fingerprinting analysis of the material to be confirmed. 根据这一证据,检察官撤消了对嫌疑犯Z提出的刑事诉控。 Based on this evidence, the prosecutor withdrew the proposed criminal suspects Z v control.

实施例4集合的小随体探针鉴于这些小随体在Southern印迹杂交中的敏感性,从而可将探针池(集合物)用于自同时存在的几个位点检出可变DNA片段,图5中显示了使用包含5个探针(pλg3、λMS1、λMS8、λMS31和λMS43)的探针池检测之结果。 4 minisatellite probe sets embodiment in view of the sensitivity of these minisatellites in Southern blot hybridization, probe pool thereby (bulk material) is used since several sites simultaneously present variable DNA fragments detected Figure 5 shows the results of detection of the probe pools containing 5 probes (pλg3, λMS1, λMS8, λMS31 and λMS43) in. 理论上,可被5个探针检出之不同DNA片段的数目是&Sigma;15]]>(1+Hi),其中Hi是第i个探针的杂合性。 In theory, the number of different DNA fragments can be detected in 5 of the probe is & Sigma; 15]]> (1 + Hi), where Hi is the i-th probe heterozygosity. 就这5个探针来说,每个人应平均被检出9.78个片段。 In this five probes, each person should be checked out an average of 9.78 fragments. 实践中,则可分辨出8.4±1.2(SD)条大于2Kb的带(被试者为40个随机选择的个体)。 In practice, can be distinguished 8.4 ± 1.2 (SD) shall be greater than 2Kb band (The subjects were 40 randomly selected individuals). 这种丢失可分辨带的情况部分原因是由于排除了小的(1.6Kb)和相对常见的(共有的)pλg3等位基因(实施例1,每一个体平均丢失0.33条带),但更主要的还是由于不同的小随体等位基因的电泳共迁移。 This loss of resolvable bands case partly due to exclusion of the small (1.6Kb) and relatively common (shared) pλg3 allele (Example 1, an average loss of 0.33 each individual band), but mostly or because of different minisatellite alleles electrophoretic co-migration.

多位点Southern印迹图形是高度个体特异的,只有18%(SD±11%)的DNA片段是由随机选择之北欧人个体对之间共享的(试验了40对)。 Multilocus Southern blot graphics are highly specific for the individual, and only 18% (SD ± 11%) of DNA fragments between randomly-selected individuals by Nordic share (40 pairs tested). 同胞之间,共享之片段的水平增到~57%(试验了10对)。 Among compatriots level of fragments share increased to ~ 57% (tested 10 pairs).

在父/母/子三元组中,所有子代的片段均可追踪到父母亲(图12)。 The father / mother / child triples, all offspring fragments can be traced to the parents (Figure 12). 每个子代都含有3.4±0.5(SD)个属父系起源的DNA片段,以及同等数目之母系特异的DNA片段(试验了10个父/母/子三元组)。 Each offspring contains 3.4 ± 0.5 (SD) genera paternal origin of the DNA fragment, and an equal number of maternal-specific DNA fragments (tested 10 father / mother / child triplets).

如上属所指出的,图12显示了经与合并的小随体探针杂交,可检测出人DNA中的多个高可变位点。 As noted in the case of Figure 12 shows by minisatellite probe hybridization with the merger, can detect multiple hypervariable loci in human DNA. 用HinfⅠ酶切4μg得自随机选择的英国人(1-6)、同胞对(7、8和9、10)以及父/子/母家庭组(11、12、13和14、15、16)的样品,通过0.8%琼脂糖凝胶作电泳分离,直到所有小于2Kb的片段都从凝胶中泳出。 HinfⅠ digested with 4μg from randomly selected British (1-6), sib pairs (7, 8 and 9, 10), and parent / child / parent family group (11,12,13 and 14,15,16) sample, by 0.8% agarose gel electrophoresis for separation, until all fragments are less than 2Kb swimming from the gel. 将酶解产物与来自pλg3、λMS1、λMS8、λMS31和λMS43的各2ng DNA插入片段作Southern印迹杂交;在借助随机寡核苷酸引发法用32P标记之前,合并各插入片段。 The hydrolyzate and from pλg3, λMS1, λMS8, λMS31 and λMS43 each 2ng DNA insert fragment as Southern blot hybridization; initiator in the use of random oligonucleotide method using 32P-labeled before combined each insert. 带之标记强度的变异可能是因个别探针的标记和杂交效率不同所导致的。 The band labeling intensity variation may be due to different efficiency of labeling and hybridization of individual probes caused.

Classifications
International ClassificationG01N33/50, C07H21/00, C12N15/10, C12N15/00, C12N15/09, C12Q1/68
Cooperative ClassificationC12Q1/6827, C12Q1/6876, C12N15/10, C12Q2600/172, C12Q2600/156
European ClassificationC12N15/10, C12Q1/68B6, C12Q1/68M
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