WO1989001979A1 - Sonde d'adn - Google Patents

Sonde d'adn Download PDF

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Publication number
WO1989001979A1
WO1989001979A1 PCT/GB1988/000685 GB8800685W WO8901979A1 WO 1989001979 A1 WO1989001979 A1 WO 1989001979A1 GB 8800685 W GB8800685 W GB 8800685W WO 8901979 A1 WO8901979 A1 WO 8901979A1
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WIPO (PCT)
Prior art keywords
sequence
dna
restriction
polynucleotide
probe
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PCT/GB1988/000685
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English (en)
Inventor
Ian Craig
Neil Fraser
Yvonne L. Boyd
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Isis Innovation Limited
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Publication date
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Publication of WO1989001979A1 publication Critical patent/WO1989001979A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • hypervariable probe which detects differences in individuals at the DNA level (5).
  • the hypervariable probe is able to detect variable DNA sequences in human DNA.
  • the hypervariable regions detected by this probe comprise a basic DNA sequence repeated a number of times. Hypervariable regions have been detected at different places on many chromosomes. The variability of these regions is due to differences in the number of times the repeat sequence of a region is repeated.
  • DNA fragments which contain iiypervariable regions.
  • DNA fragments can be separated by well known means according to the number of base pairs in a fragment. In the case of the hypervariable regions the size of a fragment produced will depend on the number of times the repeat sequence is repeated in that region thus giving restriction fragment length polymorphisms.
  • the fragments can be detected by hybridisation with the hypervariable probe. These can be displayed as "genetic fingerprints" by simple examination of DNA prepared from extremely small samples of body tissue. Such is the extent of the variability detected by the probe that only identical twins have undistinguishable patterns.
  • the hypervariable probe, and derivatives from it which can identify variable regions on particular human chromosomes can be used to map inherited diseases and to follow them through families Inheritance or tne fragments has been shown to be Mendelian. Using these probes it is possible to predict the likely disease status of progeny.
  • variable regions detected by the hypervariability probe(5) are all autosomal in origin. No X-chromosome linked sequences have hitherto been detected.
  • a DNA sequence has now been isolated independently and without reference to previously described variable sequences. This novel sequence hybridises to an X-chromosomal hypervariable region and flanking sequences.
  • the present invention provides a
  • Xp11.3 region that has the pattern of restriction sites generally as shown in figure 1.
  • It contains a region which comprises a variable number of repeats of a repeat sequence (HYPERXOX) which, when X chromosomal human DNA is cut with a restriction enzyme that does not cut within the repeat sequence, produces a restriction fragment whose length is variable between one individual and another depending en the number of times the repeat sequence is present.
  • HYPERXOX a repeat sequence which, when X chromosomal human DNA is cut with a restriction enzyme that does not cut within the repeat sequence, produces a restriction fragment whose length is variable between one individual and another depending en the number of times the repeat sequence is present.
  • Figure 1 is a restriction map o f the cosmid
  • Figure 2 is the sequence of a Sau 3A/HaeIII restriction fragment isolated from the sequence M27 which includes 31 ⁇ 2 copies of a 26 bp repeat sequence HYPERXOX.
  • Figure 3 is a southern blot of EcoRI digested female DNAs probed with M27 ⁇ demonstrating multiallelic variation.
  • Figure 4 is a southern blot of EcoRI digested DNA from related individuals probed with M27 ⁇ which shows simple sex linked Mendelian inheritance.
  • Figure 5 and 6 are pedigrees and EcoRI digests of DNA from families in which the disease retinitis pigmentosa is known to occur.
  • Figures 7 and 8 a re Msp1 and Hpall digests of X chromosomal DNA probed with M27 ⁇ illustrating the pattern of X inactivation.
  • Figure 9 is a proposed structure for the repeating sequence HYPERXOX.
  • the sequence comprises the DNA fragment of the M27 ⁇ region including HYPERXOX.
  • Single stranded DNA probes labelled with a detectable marker are conventionally prepared DNA molecules or fragments thereof for use in detecting complementary sequences.
  • the present invention provides a DNA probe comprising single stranded DNA as defined. which single stranded DNA molecule is labelled with a detectable marker.
  • the detectable marker will be selected from fluorescent and radioactive markers and components of chromogenic and chemiluminescent enzyme systems. Such markers are well known in the art.
  • the sub-regional localisation of M27 ⁇ to the pericentric region of the X chromesome means that the probe may be of use for linkage analysis of disease loci thought to reside in this region. Because the level of recombination between sequences near to the centromere is thought to be lower than that between sequences located more distal ly it may be applicable to loci on proximal Xq as well as those on proximal Xp. Loci in this region include those for retinitis pigmentosa (Xp11.3). incontinentia pigmenti
  • Xp11 Menkes syndrome
  • Xp11-q11 Menkes syndrome
  • Xp11-q12 Wiskott-Aldrich syndrome
  • the present invention provides an assay method (suitable inter alia for determining the inheritance of hypervariable regions within families and the likely disease status of progeny) which comprises treating X-chromosomal DNA which has been cut with a restriction enzyme with the DNA probe of the invention under hybridising conditions then analysing the treated DNA to detect the presence and/or size of fragments in an individuals DNA.
  • an assay method (suitable inter alia for determining the inheritance of hypervariable regions within families and the likely disease status of progeny) which comprises treating X-chromosomal DNA which has been cut with a restriction enzyme with the DNA probe of the invention under hybridising conditions then analysing the treated DNA to detect the presence and/or size of fragments in an individuals DNA.
  • the DNA in the gel is transferred to a nitrocellulose filter and is there hybridised with the labelled probe which binds to any DNA fragments containing a DNA sequence complementary to the probe. Any bound probe is then detected by means of its detectable marker.
  • a DNA fragment will be produced for each X-chromosome. Thus, two fragments will normally be produced by female DNA and one fragment will normally be produced by male DNA. Comparison of positions of fragments on gels from family members will reveal which of the X fragments was inherited by progeny, and the likely disease status of progeny with respect to X-chromosome linked diseases with loci mapping in the region of the probe.
  • a further aspect of the invention provides a method for the screening and diagnosis of disease states in which an abnormal number of X-chromosomes are present.
  • One way of carrying out such a method would be to employ the Southern blotting technique. In such a method the number of fragments of different length produced is normally the same as the number of X-chromosomes present. Thus simply determining the number of fragments present would enable diagnosis to be made.
  • the probe has been employed to demonstrate the maternal contribution of both X chromosomes in a case of Klinefelter's syndrome.
  • the invention also provides a method for studying the inactivation of X chromosomes in cells from a wide variety of sources.
  • the enzyme Msp1 cleaves the sequence CCGG and will do so even when the internal cytosine is methylated; its isoschizomer Hpall will only cleave at an unmethy lated CCGG sequence.
  • Probes of this invention and the hypervariable region do not contain target sites for these enzymes thus hypervariability extends to fragments generated by them. It has been observed that the active X chromosome is methylated and the inactive X unmethylated at target sites adjacent to binding sites of probes of this invention. Preliminary studies confirm predictions based on results with other enzymes ie that most females will be heterozygous for Msp1/HpaII restriction fragment alleles.
  • Probes according to the present invention may be employed to assess the X inactivation status in many situations eg imprinting, X-linked disorders in females and clonal analysis - through the persistence of one pattern of methylation resulting from the consistent inactivation of one of the two X-chromosomes in the descendants of a single somatic cell. This type of approach has been described for other (non- hypervariable) X-chromosome probes (7).
  • the DNA sequence characterised above was isolated by the following procedure.
  • the hybrid MOG-T has two fragments of the human X-chromosome translocated onto mouse chromosomes spanning the whole of the X-chromosome as its only human ccmponent (2). Together the fragments a re thought to span the whole of the X chromosome.
  • the X localisation and sub-regional assignment of M27 ⁇ was achieved using a hybrid mapping panel consisting of Eco RI digested DNA from hybrid cell- lines containing different representations of the human X-chromosome.
  • the hybrids used in this study were:
  • a restriction map of the cosmid M27 has been constructed in the region of M27 ⁇ (see Fig.1). Restriction fragments to the left of the Bgl II site B + (constant region) detected in genomic digests probed with M27 ⁇ (HXoxS1) a re the same size as those present in the cosmid. Genomic restriction fragments to the right of B + (Bgl II-Eco RI, Bgl Il-Bgl II, Bgl II - PvuII and Bgl II- Hind III) detected by M27s and HXoxS2 "show pronounced variation between different individuals (variable region).
  • the restriction fragment B + -E + (HXoxS2) appears to contain a deletion of 2.8kb, since the Bgl II-Eco RI genomic fragment detected by HXoxS2 in the hybrid MOG-T is ⁇ 3.6kb a s opposed to the O.Skb predicted from this map. Many of the repeats responsible for the observed hypervariability are presumed to fall within this deletion. A plausible explanation for the observed deletion of sequences is that the repeat responsible for the hypervariability at this locus is unstable in the recA + E. coli host used to propagate the cosmid library (E.coli ED8767). This phenomenon has been noticed by several other workers. In an attempt to isolate this repeat an approach described by Nicholls et al. (6) has been followed.
  • the probe comprises a DNA fragment present in multiple copies within the hypervariable repeat region.
  • the hypervariable repeat HYPERXOX is the sequence that is responsible for the observed multiallelic variation.
  • the sequence described as M27 ⁇ refers to the mainly single-copy probe that detects hypervariability but which also includes a number of repeats of the repeat sequence HYPERXOX.
  • a Sau3A/HaeIII restriction fragment has been isolated from M27 ⁇ which contains three complete and one partial copy of a 26 base pair repeated sequence.
  • the complete sequence of this restriction fragment is provided in Figure 2.
  • the repeating motif is designated in bold lettering. It can be seen ( Figure 9) that the 26 base pair repeat sequence HYPERXOX contains within it a perfect inverted repeat of 10 base pairs separated by 3 base pairs and has therefore the potential to generate a cruciform structure with a symmetrical stem loop configuration.
  • This type of sequence may be of biological significance particularly in promoting recombination. It is possible that such a sequence is distributed elsewhere in the human genome and in the genomes of other organisms.
  • a further derivative of the 26 bp repeat sequence HYPERXOX has been prepared by ol igonucleotide synthesis and comprises the polynucleotide
  • oligoHYPERXOX is incapable of forming a stable internally folded structure and, unlike HYPERXOX, can be easily hybridised to complementary sequences.
  • Radioacti vely labelled oligoHYPERXOX is capable of hybridising to the restriction fragment carrying HYPERXOX repeats in digests of M27 ⁇ .
  • Probes comprising the polynucleotide sequences oligoHYPERXOX and HYPERXOX or complementary sequences will bybridise to the HYPERXOX or its complementary sequence and to DNA fragments which include the repeat sequence.
  • Other probes comprising sequences such as oligoHYPERXOX which include a significant part of the HYPERXOX sequence or complementary sequence will also hybridise to DNA fragments containing the repeat sequence provided that a sufficient part of the sequence is used to ensure specific hybridisation.
  • a probe comprising a polynucleotide sequence having a sufficiently high degree of homology with the HYPERXOX sequence or complementary sequence or a significant part tnereof to give a specific pattern of hybridisation could be used to detect DNA fragments containing the repeat sequence.
  • polynucleotide probes suitable for detecting the variable DNA fragments containing the HYPERXOX can be prepared from any polynucleotide sequences which hybridise specifically to the HYPERXOX sequence or its complementary sequence. The shorter the sequence or the lower the homology the less specific the hybridisation will be. Using such less specific probes, it may be possible to isolate sequences related to HYPERXOX from throughout the human genome or genomes of other species. The degree of homology should not however be reduced so far as to cause binding to unrelated sequences.
  • probes comprising the polynucleotide sequence of the single copy sequence flanking the HYPERXOX repeat sequence as shown in Figure 2 will hybridise to restriction fragments containing the HYPERXOX sequence. Again a probe comprising only sufficient of the sequence, or complementary sequence, to give specific hybridisation could be used rather than the whole sequence.
  • HYPERXOX sequence it can be used as a probe to isolate further sequences which bind to the single copy regions flanking the HYPERXOX region any of which can be used as a probe to detect hypervariability due to HYPERXOX provided that the restriction enzyme used does not cleave between the hybridisation site of the probe and the HYPERXOX region or within the HYPERXOX sequence.
  • the probe M27 ⁇ can be isolated by synthesising a polynucleotide probe including at least part of the sequence complementary to that of the Sau3A/HaeIII fragment give in Figure 2 . The probe so produced is then used to probe an EcoRI digested genomic library constructed from the MOG-T hybrid cell line under hybridising conditions as previously described.
  • the present invention thus also provides a method of making probes hybridising to restriction fragments containing a variable number of copies of the repeat sequence HYPERXOX which method comprises the following steps;
  • the plasmid M27 can form the basis for the production of a reagent containing as its significant elements (1) a gene conferring a selectable phenotype following its introduction into target cells eg neomycin resistance (2) copies of the HYPERXOX sequence flanking the selectable "marker” gene (3) sequences enabling the propagation and selection of the modified plasmid in bacteria.
  • This reagent can be introduced into target cells by the process known as "transfection”; a procedure described in "A Practical Guide to Molecular Cloning" (Perbal, B. 1984 Wiley Interscience pp510-512). Following “transfection", a proportion of donor sequences will be integrated into the recipient genome through recombination.
  • Homologous recombination occurs at low frequency, but may be considerably enhanced through the HYPERXOX sequences. Such a procedure could serve to introduce a selectable marker into the genome at HYPERXOX sites. Further enrichment of the adjacent sequences, including linked genes, could be achieved by the established procedures of chromosome mediated gene transfer (CM6T) and preparation of genomic libraries (see eg. Pritchard, C. and Goodfellow, P.N. 1987 Development 101, Supplement 59-65). Furthermore, the possible enhancement of homologous recombination via HYPERXOX would allow the introduction of other genetic material into the genome of humans and other organisms. In particular, this approach would enable the introduction of a normal gene copy into a recipient with a defective gene (gene therapy) or the addition of a new gene into an experimental organism (gene manipulation).
  • the hypervariabi 1 ity detected by the probe and the localisation to Xcen-Xp11.3 suggests that the marker will be of considerable value in mapping and in prenatal diagnosis of diseases whose loci map to this region (retinitis pigmentosa and incontentia pigmenti) and in the analysis of other disease loci believed to be pericentrically located including Mehke's syndrome.
  • Retinitis pigmentosa occurs at a frequency in the region of 1-2 in 5,000 of the general population 36-38% of RP patients are isolated cases, the remainder show autosomal dominant, autosomal recessive or X- linked modes of inheritance.
  • the X-linked variety (XLRP) is found in 14-22% of RP families in the U.K.
  • Female 49 is an obligate carrier, because she has had an affected son and daughters that have had affected sons in subsequent generations. She has alieles of
  • Probe DNA was prepared from blood (B), lymphoblastoid cell lines (L) or somatic cell hybrids (H) by standard methods. 5ug samples were digested with Msp1, or Hpall using restriction buffers supplied by the manufacturer (BRL), electrophoresed and transferred to Hybond N nylon membrane (Amersham Int.). Probe DNA was labelled by nick-translation to a specific activity of 10 dpm/ug and hybridisations and washes carried out to a final stringency of 0.5X SSC at 63°C. Details of Samples Studied
  • Figure 7 normal males whose X chromosome is therefore active show a single band with Msp1 digests since Msp1 cleaves irrespective of methylation of the internal cytosine.
  • Hpall digests the single band is replaced by high molecular weight material. Hpall does not cleave when the internal cytosine is methylated.
  • Normal females show two bands with Msp1 digests. After digestion with Hpall both bands are present but at reduced intensity and accompanied by high molecular weight material. This confirms most females are heterozygous for X in activation.
  • Figure 8 - females with defined patterns of X inactivation show bands present in Msp1 digests that are absent from Hpall digest and high molecular weight material is present. The presence of the other band(s) in both digests provides a good control for the Hpall digestion.
  • Somatic cell hybrids containing M27 ⁇ locus on an active X chromosomel give the same pattern as males.

Abstract

Sont décrites une région hypervariable chromosomique X, ainsi que des sondes d'ADN adaptées pour détecter des fragments d'ADN, comprenant la région hypervariable. L'utilisation des sondes comprend l'analyse du statut d'activation du chromosome X, la possibilité de clonage et le diagnostic de maladies liées à l'X.
PCT/GB1988/000685 1987-09-04 1988-08-19 Sonde d'adn WO1989001979A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB878720855A GB8720855D0 (en) 1987-09-04 1987-09-04 Probe
GB8720855 1987-09-04

Publications (1)

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WO1989001979A1 true WO1989001979A1 (fr) 1989-03-09

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EP (1) EP0377627A1 (fr)
JP (1) JPH03501202A (fr)
GB (1) GB8720855D0 (fr)
WO (1) WO1989001979A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178220A2 (fr) * 1984-10-01 1986-04-16 Institut Pasteur Vecteur rétroviral
EP0186271A1 (fr) * 1984-11-12 1986-07-02 THE LISTER INSTITUTE OF PREVENTIVE MEDICINE Royal National Orthopaedic Hospital Méthode de caractérisation d'un échantillon d'ADN génomique
EP0238329A2 (fr) * 1986-03-19 1987-09-23 Zeneca Limited Sondes génétiques
WO1988000979A1 (fr) * 1986-07-25 1988-02-11 The Children's Medical Center Corporation Sondes dmd
EP0266787A2 (fr) * 1986-11-07 1988-05-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé pour la détection des polymorphismes de restriction-longueur de fragment dans les génomes eucaryotiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0178220A2 (fr) * 1984-10-01 1986-04-16 Institut Pasteur Vecteur rétroviral
EP0186271A1 (fr) * 1984-11-12 1986-07-02 THE LISTER INSTITUTE OF PREVENTIVE MEDICINE Royal National Orthopaedic Hospital Méthode de caractérisation d'un échantillon d'ADN génomique
EP0238329A2 (fr) * 1986-03-19 1987-09-23 Zeneca Limited Sondes génétiques
WO1988000979A1 (fr) * 1986-07-25 1988-02-11 The Children's Medical Center Corporation Sondes dmd
EP0266787A2 (fr) * 1986-11-07 1988-05-11 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé pour la détection des polymorphismes de restriction-longueur de fragment dans les génomes eucaryotiques

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JPH03501202A (ja) 1991-03-22
GB8720855D0 (en) 1987-10-14
EP0377627A1 (fr) 1990-07-18

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