CA2194697A1 - Method, reagents and kit for diagnosis and targeted screening for p53 mutations - Google Patents
Method, reagents and kit for diagnosis and targeted screening for p53 mutationsInfo
- Publication number
- CA2194697A1 CA2194697A1 CA002194697A CA2194697A CA2194697A1 CA 2194697 A1 CA2194697 A1 CA 2194697A1 CA 002194697 A CA002194697 A CA 002194697A CA 2194697 A CA2194697 A CA 2194697A CA 2194697 A1 CA2194697 A1 CA 2194697A1
- Authority
- CA
- Canada
- Prior art keywords
- seq
- gene
- exon
- exons
- mutation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
Rapid and cost effective diagnosis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost: (a) immunoassays; (b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene. Preferably, the amplification primers are multiplexed so that more than one DNA fragment is amplified in a single vessel, using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal p53 gene; and (c) analysis of DNA from a patient sample by DNA sequencing of the p53 gene beginning with the sequencing of those regions most likely to harbor point mutations, and proceeding to sequence regions less likely to harbor point mutations.
Description
~WO 96101909 I ~
METHOD, REAGENTS AND KIT FOR DIAGNOSIS AND
TARGETED S~RT~NTN~ FOR p53 ~UTATIONS
This application is a continuation-in-part of co-pending U.S. Patent Application No. 08/271,946, filed July 8, 1994, which is incorporated herein by reference.
p2,~ RflTT~I OF TT-TP lNVl",N'l'lUN
This application relates to a method, reagents and kit for diagnosis and targeted screening of mutation in p53 protein and mutation in the gene coding for the p53 protein (herein the "p53 gene~l Such ~utations are collectively referred to herein as ~p53 mutations~.
The evidence for the tumor suppressor activity of wild-type p53 is now extensivc. Since its discovery in 1979 when it was found to he ~n~plr-YPfl with the SV40 large T
antigen in SV40-transformed rodent cells the significance of p53 has slowly come to light. The protein appears to act as a transcription factor, and may be responsible for apoptosis of pre-cancerous cells. (Ziegler et al., ~Sunburn and p53 in the onset of skin cancer~, Nature 372: 773-776 ~1994~) The DNA se~uence of the 11 exons of the p53 gene is now known and close to 1000 papers were published on p53 in 1993.
P53 mutations are significant because they are found in an Pnr- q variety of tumors. Among common tumors, about 70~ of colorectal cancers, 50% of lung cancers, and up to 40% of breast cancers carry p53 gene mutations. p53 is also linked to cancers of the blood and lymph nodes, including Hodgkin~s disease, T cell lymphoma and certain kinds of leukemia. Moreover, aberrant forms of the p53 gene are correlated with more aggressive tumors, metastasis and lower 5-year survival rates. Such reports have emerged for cancers of the colon, lung, cervix, bladder, prostate, breast and skin.
The serious consequences of p53 mutations mandates a method for detection and diagnosis of such mutations which _ _ _ _ _ _ _ _ _ . _ . . ...
METHOD, REAGENTS AND KIT FOR DIAGNOSIS AND
TARGETED S~RT~NTN~ FOR p53 ~UTATIONS
This application is a continuation-in-part of co-pending U.S. Patent Application No. 08/271,946, filed July 8, 1994, which is incorporated herein by reference.
p2,~ RflTT~I OF TT-TP lNVl",N'l'lUN
This application relates to a method, reagents and kit for diagnosis and targeted screening of mutation in p53 protein and mutation in the gene coding for the p53 protein (herein the "p53 gene~l Such ~utations are collectively referred to herein as ~p53 mutations~.
The evidence for the tumor suppressor activity of wild-type p53 is now extensivc. Since its discovery in 1979 when it was found to he ~n~plr-YPfl with the SV40 large T
antigen in SV40-transformed rodent cells the significance of p53 has slowly come to light. The protein appears to act as a transcription factor, and may be responsible for apoptosis of pre-cancerous cells. (Ziegler et al., ~Sunburn and p53 in the onset of skin cancer~, Nature 372: 773-776 ~1994~) The DNA se~uence of the 11 exons of the p53 gene is now known and close to 1000 papers were published on p53 in 1993.
P53 mutations are significant because they are found in an Pnr- q variety of tumors. Among common tumors, about 70~ of colorectal cancers, 50% of lung cancers, and up to 40% of breast cancers carry p53 gene mutations. p53 is also linked to cancers of the blood and lymph nodes, including Hodgkin~s disease, T cell lymphoma and certain kinds of leukemia. Moreover, aberrant forms of the p53 gene are correlated with more aggressive tumors, metastasis and lower 5-year survival rates. Such reports have emerged for cancers of the colon, lung, cervix, bladder, prostate, breast and skin.
The serious consequences of p53 mutations mandates a method for detection and diagnosis of such mutations which _ _ _ _ _ _ _ _ _ . _ . . ...
earliest stage of tumor development. Toward this end, ;mmnnn~qSayS and DNA assays for p53 mutations are known in the art. To date, however, neither method has been able to identify p53 mutations with a hiqh degree of specificity and accuracy in a cost effective fashi-on. Those irnmuno-assays that have been published identify only a small portion of those patierts actually thought to be carrying the mutation.
Two general techniques of immunoassay have been employed. The first technique is an indirect method which detects anti-p53 antibodies that arise in some patients who have p53 mutations. Irnmunoassay tests for detecting anti-p53 antibodies in patient sera are currentl~ based on radio-active labeling, immunoprecipitation and immunoblotting.
All these methods are qualitative and time consuming and thus not suitable for screening larqe number of samples.
~Angelopoulou et al., ''Autoantibodies against the p53 tumor suppressor gene product quantified in cancer patient serum with time-resolved immunofluorometry~, Cancer J 6(6): 3i5-321 ~1993)).
The second technique of immunoassay directly detects mutant p53 protein. Such methods have been disclosed in at least two publications. sartek et al. disclosed an enzyme-linked immunosorbent assay for p53 which was applied for the measurernent of p53 in tumor tissue extracts (Oncogene, 6:
1699-1703 (l991)). ~assapoglidou et al developed a mono-clonal antibody useful for direct detection of mutant p53.
(Oncogene 8: 1501 1509 (1993).) DNA analysis of p53 rnutat:ions has been reported using various techniques Sinqle stranded conformational poly-morphism was used to detect mutant p53 by Ruypers et al.
("Detection of point mutations in DNA using capillary electrophoresis in a polymer network", J. Ckromat-ography 621:
14g-56 (1993j), Runnebaum et al. (NMutations in p53 as potential molecular markers for human breast cancer" Proc.
Nat~l Acad. Scl. USA 88: 10657-10661 ~1993) and by Felix et al ("Absence of hereditary p53 mutations in 10 familial AM~EN~ED SHEET
~ 1 94~
leukemia pedigrees", J Clin Invest 90: 653-8 (1992)). Actual genomic DNA sequencing diagno~,is was performed on relatively small groups of patients by Toguchida et al. ("Prevalence and spectrum of germline mutations of the pS3 gene among S patients with sarcoma'', N Engl J ~ed 326: 1301-8 (1992)) and by Malkin et al. ("Germline mutations of the pS3 tumor-suppressor gene in children and young adults with second malignant neoplasms', N En~l J ~.e~ 326: 1309-lS
(1992)). Detection of p53 mutations is also discussed in EP-A 0 390 323. The cDNA sequence of a larger group of patients (over 400) was reported in a Pharmacia LKs meeting publication (Andell et al. "A new approach in automated DNA
sequencing to analyse the pS3 gene in a large nu7nber of breast cancer patients", Pharmacia LKB meeting literature, lS 1994) These rapid DNA-based techniques have been used to detect mutations, but because they are so labor intensive, that large-scale screening tests are impractical (Harris et al., "Clinical implication.s of the pS3 tumor-suppressor gene', N En~1 J ~ed 329:1318-1327 (1993)).
Thus, the existing methods of diagnosis have been frustratingly unsatisfactory. Researchers have used either immunoassay or DNA analytical methods to diagnose pS3 mutation, even though such tests result in numerous false negatives. A method is required for rapid and cost effective diagnosis of p53 mutation in the millions of individuals who develop potentially lif.e threatening malignancies each year. Dunn et al., J. Ce71 sioche~ ~ol Suppl 18C: 199 (1994) briefly describes an assay system for Rsl mutations which is said to be cost effective, but does not describe such a system for pS3.
It is an object of this invention to provide a method for rapid and cost effective diagnosis of p53 mutations in a . sample of patients.
It is~a further object o~ this invention to provide DNA
se~uencing and amplification ~rimers specific for analysis of the pS3 gene from a patient sample.
AMENDE~ SHEET
2 1 9 4 ~I r 7f It is a further object of this invention to provide kits of DNA oligonucleotides for amplification and seq~encin~ of the p53 ~ene of a patient.
It is a further object o~ this in~ention to provide a method of generatin~ a pS3 mutation report which is used to AMENDED SIIEET
2 1 9'~ 7 provide appropriate genetic counseling to the patient and family upon whom the test i5 performed.
SUMM~RY OF T~ lNv~ s~ UN
Ir. accordance with the present invention, rapid and cost effective ~iA~nnsis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost:
~a) ; ~ACSays~ particularlY i naqsays for anti-p53 antibodies present in a patient sample;
lS ~b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene. Preferably, the amplification primers are mul~;~F~ so that more than one DNA fragment is amplified in a single vessel, using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal p53 gene; and ~c) analysis of DNA from a patiert sample by DNA
sequencing of the p53 gene beginning with the sequencing of those regions most likely to harbor point mutations, and proceeding to sequence regions less likely to harbor point mutations.
The present invention includes a multitude of amplifi-cation and sequencing primers. These primers, taken indivi-dually or as part of kits for the detection of p53 mutations represent a further aspect of the present invention.
Particularly preferred primers constitute sets that are compatible for coampli~ication and that produce amplified DNA fragments of distinctive lengths from other fragments amplified in the same set.
2~ ~fi~
. . ~ .
- 5 - .
test is performed. The generation of such reports, which may be in the form of a printed report, an electronic communication, such as a facsimile or electronic mail (e-mail~ transmission, or a posting of a data entry in a cornputer record relating to the patierlt~ is a further aspect ~ of the present invention.
PRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the hierarchy of p53 diagnosis as provided by the invention; and Fig. 2 outlines two methods of imrnunoassay for the presence of anti-p53 antibodies in patient sera.
DETAILED DESCRIPTI:ON OF THE INVE~TION
The present invention reLates to a method for the identification of p53 mutations, a method for generating reports for providing counsellng to patients and families of patients with p53 mutations, and to oligonucleotide primers and kits useful in practici~g these methods. The present invention utilizes the hierar:hical approach which is disclosed generally in US Patent Application Serial No.
08/271,946, and in a concurrently filed continuation-in-part thereof, PCT~US95~08606, (Attorney Docket No. VGEN.P-002-WO~
which is incorporated herein by reference.
The method for identification of p53 mutations in a sample of patients is based upon using diagnostic tools 1~
in a hierarchy of increasing accuracy and cost per tool; and 2~ in which each tool is of extremely high specificity and detects essentially no false positives. A sample which exhibits a positive result establishes the presence of a p53 mutation and a patient report can be prepared on this basis.
A sample which exhibits a negative result is thereafter --subjected to a more costly, but more accurate test to determine if a mutation is present.
Thus, as shown in Fig. 1, an example of such a hierarchy comprises, in order, an immunoassay step; a DNA
fragment length~quantity analysis; and DNA sequencing. In A~IIENDED SHEET
_ _ . _ _ _ . . .. ... . .. . .... .. . .
2 i ~4fiq7 W096/01909 r~.~.,.
~, Thus, as shown in Fig. 1, an example of such a hierarchy comprises, in order, an i oACSay step; a DNA
fragment length~quantity analysis; and DNA seguencing. In the exemplary results shown in Fig. 1, the first step has the advantage of low cost, but detects a positive result in only 15% of the patients. The second level of the test hierarchy is of moderate cost, but because of its increased accuracy it detects a positive result in 30~ (26 of 85) of the patients who tested negative using the ; --csay~
This means that the more expensive DNA Seguencing analysis need only be performed on 59% of the original patients.
Thus, the use of the hierarchical approach leads to a substantial reduction in the average cost of the test while providing high levels of accuracy for every patient, by having eliminated from the test pool those samples that are known to be positive.
TMMTlNf)~.~sAy pRo~n~
As illustrated in Pigure 1, the first level in the hierarchy of p53 diagnosis may be an ; -~csay. Because of the relatively low cost of i nAcsay~ it may be advantageously used to eliminate a significant number of patients from the sample pool prior to proceeding to the next level of the hierarchy. Methods of i nAqsay that can be employed at this first stage have been discovered by many researchers. The following list of papers all include methods for identifying p53 mutations by i -~Csay, though by no means is this list exclusive, and a practitioner skilled in the art may know of alternative methods of p53 ; nAcSay Angelopoulou et al., NAutoantibodies against the p53 tumor suppressor gene product ~uantified in cancer patient serum with time-resolved immunofluorometry~, Cancer ~ 6(6):
315-321 (19g3).
Bartek et al., Oncogene 6: 1699-1703 (1991~.
~ 1 q ~
~ W096lol909 ~ r~
_ - 7 -Crawford et al., "Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer~, Int J Cancer 30: 403-8 (1982).
~ Caron De Froentel et al., Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell lymphomaN, Int J Cancer 39: 185-9 ~1987).
Christopoulos et al., ~Oncogenes and tumor suppressor genes: new biochemical tests.~ CRC Cri t Rev Clin Lab Sci 29: 269-305 (1992).
Davidoff et al., UImmune response to p53 is fl~p~nflPnt upon p53 HSP70 complexes in breast cancers", Proc Natl Acad Sci USA 89: 3439-42 ~1992).
Hassapoglidou et al., ~Antibodies to the p53 tumor suppressor gene product quantified in cancer patient serum with a time-resolved immunofluorometric technique~, Clin Biochem 25:445-9 (1992).
Hassapoglidou et al., ~Quantification of p53 protein in tumor cell lines, breast tissue extracts and serum with time-resolved immunofluorometry , Oncogene 8:1501-1509 (1993).
~abrecque et al., ~Analysis of the anti-p53 antibody response in cancer patients~, Cancer Res 53: 3468-71 (1993).
Schlichtholz et al., ~he immune response to p53 in breast cancer patients is directed against i ofl~ n~nt epitopes unrelated to the mutational hot spot~, Cancer Res 52: 6380-84 (1992).
Volkmann et al., ~The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for ~-l;gn~ncy and in~epen~ent of the alpha-fetoprotein status~, Hepatology 18: 559-565 ~1993!
Winter et al., ~Development of antibodies against p53 in lung cancer patients appears to be dependent on the p53 mutation~, Cancer Res 52: 4168-74 (1992).
In selecting an ; o~Csay for use in the present method, it should be understood that some i ~cSayS only detect small subsets of p53 mutations, while other immuno-6 ~ ~
WOs6/0l90~ L _, ~,./{
.
assays can detect a wide variety of p53 mutations. In order to reduce the cost of diagnosis, it is advantageous to take into account the scope of detection of each ; ~s~y and select an i ~qsay which provides highly specific detec-tion for the greatest number of p53 mutations as theanalytical start point. If desired, a second ; ~csay may be used after the first immunoassay if it will identify other types of mutations not found by the first i ~.qsay.
This selection and sequential ordering of i ~qsays may continue until the patient pool is reduced to eliminate essentially all patients reasonably diagnosed with i ~qsay techniques before going on to other, more expensive and more accurate assays.
DNA ANATYSIS
The next level in the hierarchy of this invention test those patient samples which did not prove positi~e for p53 mutation based on i ~csay, be subjected to more accurate and more costly analysis. DNA analysis of patient samples is an non-limiting example of such an analysis. Within the general class of DNA analysis, however, there is a hierarchy of methods, each level in the hierarchy providing increasing accuracy but at increased cost. At the top end of this hierarchy (most accurate and expensive~is DNA sequencing.
DNA sequencing provides the maximum degree of accuracy because each base in DNA from the sample is i~n~if;e~ and compared to the wild-type sequence.
DNA analysis also provides the option of methods which are lower in the hierarchy than DNA sequencing but which offer greater accuracy than i ~SRy pLocedu-=s. A
non-limiting example of such a test is an assay for DNA
fragment length/quantity mutations. This fragment analysis can be used as an intermediate level in a three (or more) level hierarchy, or it can be used as the first level of a hierarchy in place of the immunoassay tests described above.
FRA~MT~T ANATYSIS
As illustrated in Figure 1, level two of the hierarchy of this invention may be DNA fragment length/ quantity ~ wog6/nl9U9 ~ I 9 ¢ 6 ~ 7 ~ s 5l analysis. In this test r one or more exons of the p53 gene are quantitatively amplified using primers designed to create amplification products of known length and the lengths and/or quantity of the amplified fragments are det~rm;n~d. If there is a variance between the length of any amplified exon, and the normal length of that exon, this is an indication of an insertion or deletion mutation in that exon. The quantity of amplified material from amplifi-cation of a sample exon may also reflect the loss of genetic material. In particular, by comparing the quantity of amplified materials produced to standards amplified from a null allele (0 copies of p53), a hemizygous standard ~l copy of p53), a wild type standard (2 copies of p53) and a trisomy standard (3 copies cf p53), the nature of the mutation may be further investigated.
The number of exons tested in step two of the hierarchy is a matter of choice for the user. For example, if after repeated testing on patient samples it is found that length or quantity mutations are rarely found in certain exons, it is preferable to test these exons last, after testing other exons to see if a mutation sufficient to cause the disease is detected, before incurring the expense to test these less likely exons. In testing these other exons, the user may choose to test them one at a time, or in one multiplexing group at a time. Alternatively, the user may choose to test all exons simultaneously at once.
When a length mutation is detected in this step of the hierarchy, it is not npcecs~ry to perform additional tests on the patient sample to complete the identification process. Preferably, however, the sequence of the mutated exon will be detPnm;n~d as part of the second level of the hierarchy to confirm that the mutation detected can in fact ~ be a cause of the observed disease.
Again, all those samples that prove positive for a ~ 35 length or quantity mutation are eliminated from the patient sample, and patient reports may be made for each patient eliminated. However, if no mutation is detected at this ~o s6/o]q()~ r~
level of the hierarchy, then a more accurate and costly analysis must be undertaken. DNA sequence analysis is a non-limiting example of such a level.
~he primers usea to amplify the sample DNA for fragment analysis are oligonucleotides of defined seguence selected to hybridize selectively with particular ;oortions of the p53 gene, generally introns. Each primer has bound to it a detectable label. A preferrea example of such a label is fluorescein, which is a standard label used in nucleic acid sequencing systems usinq laser light as a detection system.
Other detectable labels can also be employed, howeYer, in-cluding other fluorophores, radio-labels, ~h~ 1 couplers such as biotin which can be detected with streptavidin-linked enzymes, and epitope tags such as digoxigenin detec-ted using antibodies available from Boehringer-~r~he;m-While considerable variation is possible in these~uence of the primers used in amplifying the exons as part of the method of the present invention, the primers used in amplification and the conditions of the amplification are preferably optimized for use in the present invention.
Looking first at the primers used, it will be understood that in order to avoid the possibility of false positiYe results the primer pair, i.e., the combination of the 5'-primer and the 3~-primer for any given e~on must be unique 2S to the p53 so that only the p53 gene will be l;fied.
~his means that the primer sequences will be generally somewhat longer than the minimum which can be used as an amplification primer. Preferred primers are from 18 to 23 nucleotides in length, without intPrn~l homology or primer-primer homology. It is also desirable for the primers toform more stable duplexes with the target DNA at the primers~ 5'-ends than at their 3'-ends, because this leads to less false priming. Stability can be appro~imated by GC
content, since GC base pairs are more stable than A~ pairs, or by nearest neighbor thermodynamic parameters. Breslauer et al., "Predicting DNA duplex stability from base sequence", Proc. ~at'1 Acad. 5ci . USA 83~ 3746-3750 ~1986~.
WO 961019U~ 0' 5''~
' 11 -In addition, to ensure complete amplification of each exon, the two primers of a pair are preferably selected to hybridize in the introns immediately flanking the exon to be amplified using the primer pair.
Additional factors apply to the selection of primers ~ for multiplexed amplification of exons, i.e., where several exons are amplified concurrently in a single reaction mix-ture. These factors are discussed in Rylchik, W., Selection of Primers for Polymerase Chain Reaction", in Methods in Nolecular Biology, Vol. 1~: PCR Protocols: Current Nethods and Applications, White, B.A. ed., Humana Press, Totowa, N.J., 1993. Briefly, applying these factors, primer pairs are selected by position, similarity oE melting temperature, internal stability, absence of internal homology or homology to each other, i.e., they won't stick to each other or to themselves, and the 3'-end will not form a stable hairpin loop back on itself.
Thus, in the present case, the goal is to have sets of primer pairs with approximately the same thermal profile, so that they can be effectively coamplified together. This goal can be achieved by having groups of primer pairs with approximately the same lenqth and the same G~C content. In addition, it is preferred that the length of the gene region between the primer binding sites on a normal p53 gene differ for each exon to be multiplexed as a group. Differences of only one base in length are sufficient, provided a high resolution gel capable of resolving one base differences is used in analyzing the amplification products. However, greater differences in length are preferred.
To evaluate compatibility of primers for use in co; ,l;fication, it is desirable to determine the predicted melting temperature for each primer. This can be accom-plished in several ways. Por example, the melting temperature, Tm can be calculated using either of the following eguations:
Tm~~C) = 81.5 + 16.6 X log [Na] + 0.41 X (%GC)- 675/length 2 ~ q4 ~
WO~61019~)9 where [Na] is the concentration of sodium ions, and the ~GC
is in number percent, or T~ (~C) = 2 X (A + T) + 4 X (G + C) where A, ~, G, and C represent the number of adenosine, thy-midine, guanosine and cytosine residues in the primer. In general, primers for coamplification should be selected to have predicted melting temperatures differing by less than 4~C.
D~ SJ:~UII~:N"~ PiT,YSI~S
Figure 1 illustrates that the final element of the hierarchy of pS3 diagnosis is DNA sequencing. DNA sequence analysis involves detPrmin;n7 the sequence of the exons to locate the mutation.
Sequencing is expensive and so it may be desirable to use a sub-hierarchy within this level of testing to reduce the 1 ik~l ih~od af having to seguence all of the exons. In this case a suitable sub-hierarchy will be det~rm;n~d by identi~ying those exons wherein mutations are most likely to occur. Mutational hotspots have been identi$ied at codons 175, 245, 248, 249, 273 and 282, which correspond to exon 5 for the first listed hotspot, exon 6 for the second three listed hotspots and exon 7 for the latter two hotspots.
(Harris CC. p53: At the Crossroads of ~olecular Carcino-genesis and Rig~ Assessment. Science 1953; 262: 1980-1981.) In accordance with this sub-hierarchy, the first exons sequenced are those which are easy to seguence and which contain hotspots. Next, if no mutation is found, hotspot exons are se~uenced which are hard to seguence (i.e. are found empirically to give less clear results when treated similarly to other exonsl. Finally, the remaining exons are sequenced in descending order of the odds of findin~ a mutation based on prior epidemiological studies. (~he order of sequencing of the exons may change as patient data acr~ tes on the location of point mutation hotspo~s.l If 6 q '~
wos~/olsos r~ ;o~
no mutation has been detected after all the exons have been sequenced, then it is concluded that there is no mutation in the test sample and a report is generated accordingly.
A preferred method for DNA sequencing as part of the method of the invention involves amplifying the exon of interest and then detPrmiring its sequence. Amplification of each exon may be performed using the same primers employ ed for fragment length analysis, although the detectable label included for the fragment analysis is not nPrPcs~ry for amplification prior to sequencing. In this case, how-ever, multiplexed amplification cannot be used since only a single amplification product is desired.
The determination of the sequence of the amplified material may be carried out in any manner. A preferred approach, however, is the ~ell-known Sanger method involving a template-dependent primer extension reaction in the pres-ence of dideoxy chain terminating nucleotides. For this method, a sequencing primer is used which hybridizes to one chain of the amplified DNA. In general, sequencing primers are nested inside the amplification primers, although the amplification primers could be used for sequencing purposes if desired.
The amplification and sequencing primers used in the present invention are advantageously packaged as kits for the detection of mutations in the p53 gene. The primers may be packaged individually within the kit, or as mixtures of primers, sometimes referred to as Nprimer cocktails,N which are useful in a single reaction vessel. Such kits may contain a single pair of primers, useful for quantitative amplification of a single exon, or multiple pairs of primers useful for amplification of multiple exons. Such kits may further include amplification and/or sequencing primers for one or more exons. Such kits may also include reagents other than primers for use in the amplification reaction, such as a polymerase and buffers, but this is optional.
Preferred kits in accordance with the invention comprise a plurality of primer pairs useful in the , .. , ... . ... . ., .. . .... _ . _ . . . .. .. . _ 2 ~ ~697 ~O g61U1909 1 ~
co amplification of a plurality of exons of the p53 qene.
Primer pairs in such kits are selected to have a common melting temperature and to produce amplification products having differing lengths.
The followins non-limiting ~Amrl~c illustrate applications of the invention.
~x~MPL~ 1 In order to determine the presence or absence of p53 mutations in a sample of patients, the following hierarchy of tests was performed.
Level 1: I rAcSav: Detection of Anti-~53 Ant;ho~ies.
The two ~ethods of Angelopoulou and DiAmAn~is ~1993, supra) were employed to detect anti-p~3 antibodies in a patient sample. Figure 2 A illustrates the principle of Method A for anti-p53 antibody quantification in patient sera. In the presence of such antibodies, a ~sandwich" is formed between a mouse monoclonal anti-p53 antibody and human anti-pS3 antibodies, resulting in high fluorescence readings. Piqure 2 ~ illustrates the principle of ~ethod s for p53 antibody quant;ficati~n in patient sera. The left-hand panel shows that in the absence of anti-p53 antibodies in human serum, the added p53 antigen is measured as shown, giving rise to fluorescence. The right-hand panel shows that when anti-p53 Ant;ho~;~p are present in patient serum, they block the added p~3 antigen, resulting in ~ow fluorescence. No 5An~wi~b can be formed between the mou8e monoclo~al and rabbit polyclonal anti-p53 ~nt;ho~;~c. ~LP=
~1~A1 ;~P phosphatase; FSAP = fluorosalicyl phosphate; FSA =
fluorosalicylate; Ab=antibody.
Instrumentation and Materials - For measuring liquid-phase Tb3~ fluorescence in white microtiter wells, a Cyberfluor 615 I ~AnAly~er time resolved fluorometer was used as described elsewhere ~Christopoulous et al., supra;
Papana tasiou-Diamandi et al., ~Ultrasensitive thyrotropin ~ nn~cs~y based on en~ymatically amplified time-resolved 21 ~46~;~
~101909 .
fluorescence with a terbium chelateN, Clin Chem 38:545-48 ~1992)). The phosphate ester of 5-fluorosalicylic acid ~FSAP) was obtained from CyberFluor Inc., Toronto, Canada.
TbCl3. 6H20 was from GfS Chemicals, Columbus OH, USA. All other chemicals were form Sigma ~h~ Al Co., St. Louis, MO, - USA unless otherwise stated.
Solutions - The enzyme substrate buffer was a 0.1 mol/L
Tris solution, pH 9.1, containing 0.1 mol NaCl and 1 mmol MgCl2 per liter. The stock FSAP substrate solution was a 2 mol/L solution in 0.1 mol/L NaOX. Fresh FSAP substrate working solutions were prepared just before use by dilution ~10-fold) of the stock in the enzyme substrate buffer. The cell lysis buffer was a 20 mmol/L Tris solution, pH 8.1, containing 150 mmol NaCl, 10 g Nonidet P-40, 0.5 mmol phenylmethylsulfonyl fluoride, 2 mg leupeptin and 2 mg aprotinin per liter. The developing solution was a 1 mol/L
Tris base solution containing 0.4 mol NaOH, 3 mmol EDTA and 2 mmol TbCl3.6X2O per liter (r.o pH adjustment). The washing solution was a 5 mmol/L Tris buffer, pH 7.80, ront~;n;n~ 0.5 g Tween 20 and 150 mmol NaCl per liter. The coating antibody solution was a 50 mr,lol/L Tris buffer, pX 7.80, containing 0.5 g sodium azide per litcr. The CM-l antibody diluent was a 50 mmol/l Tris buffer, pH 7.80. containing 60g bovine serum albumin (BSA) per liter. The goat anti-rabbit immunoglobulin alkaline phosphatase conjugate (GARRIg-ALP~ diluent was a 50 mmol/l Tris buffer, pH 7.80, containing 60g BSA, 0.5 mol KCl and 100 ml goat serum per liter.
The cell lines used in this stud~ were colon carcinoma Colo 320 HSR(+) (MnrAkAm; et al., ~Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis", Cancer Res 51: 3356-61 (1991)); pancreatic car-cinoma MIA PaCa-2 (Barton et al. ~hn~rr~l; ties of the p53 tumor suppressor gene in hum~n pancreatic cancer~, Br J
Cancer 64:1076-82 1991)): breast carcinoma T-47D (Bartek et al., ~Genetic and immunochemical analysis of mutant p53 in WO96,~l90~
human breast cancer cell lines~, Oncoge~e 5:893-9 (1990));
and human erythroleukemia OCI M2 ~Singerland et al., ~Mutations of the p53 gene in human acute myelogenous leukemia~, Blood 77: 1503-7 (1991)).
These cell lines were cultured as described elsewhere;
they all have p53 gene mutations and overproduce mutant p53 protein (~cs~roglidou et al., 1993, supra). R~cn-~;n~nt wild type p53 protein, produced as described elscwhere (Wang et al., UThe murine p53 blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T
antigenn,Cell 57: 379-92 (1989)), was a gift by Dr. Carol Prives, Columbia University, New York.
Lysates from cell lines producing p53, or recombinant p53 were diluted in a 50 mmol/L Tris buffer, pH 7.80, containing 60 g of BSA per liter for Method A and in 100 goat serum for Method B. The mouse monnrlon~l anti-p53 capture antibody (Pab240) diluent was a 50 mmol/L Tris buffer, pH 7.80, containing 60g OSA and 0.5 mol KCl per liter. Serum samples were diluted in a serum diluent which is the same as the Pab240 diluent but supplemented with 10 normal goat serum and 2~ normal mouse serum. The goat anti-human immunoglobulin-alkaline phosphatase conjugate (GAHIg-AALP) diluent was the same as the GARIg-ALP diluent.
The mouse anti-p53 - ~l~n~l antibody Pab2A0 was produced as a tissue culture supernatant from a cell line donated to us by Dr. D.P. Lane, University of Dundee, U.K.
Its antibody concentration was approximately 30 micrograms/ml. The rabbit polyclonal anti-p53 antibody, CM-1, was obtained from Di ~io~ Labs, Nississauga Ontario.
The qoat anti-rabbit and goat anti-human antibodies, conjugated to ~lk~1 ;nP phosphatase, and the goat anti-mouse antibody, Fc fragment specific (GAMIg), all approximately 1 mg/ml, were obtained from Jackson Imunoresearch, West Grove, PA.
Patient Sera - Sera from cancer patients were stored at -70 C until analysis. Sera used were ~rom patients with breast (n=105), ovarian ~n=72~, colon (n=77) and pancreatic ~ WO9~0l909 1~
- 1'1 -cancer ~n=46). For correlation studies 38 p53 antibody-positive sera from patients with the above malirnAnripc was used plus sera from prostate, lymphoma, lung and multiple myeloma patients.
Procedures:
Cell Lysis - Cells from each cell line were grown until they reached approximately lOX6 cells/ml or 90~ confluency.
The cell pellet from a 15 ml culture was lysed in 300 microliters lysis buffer, for 30 min, on ice. The cell 10 extract was centrifuged at 12,000 X g for 10 min and the pellet discarded. The lysate was used within two hours.
Total protein was measured in the lysates with the birinrhnrinic acid ~BCA) assay, commercially available from Pierce r~Pm;rAl Co., Rockford IL. Lysates typically 15 contained 1-3 mg of protein per ml.
T~~lnn~,s~y Procedure, Method A - This method is a modification of an assay previously published ~HassApog'i~nu 1992, supra). White, opaque, 12-well microtiter strips ~from Dynatech laboratories, Alexandria, VA~ were coated 20 with goat anti-mouse immunoglobulin diluted 500-fold in the coating antibody diluent ~100 microliters~ 200 ng~well, overnight incubation at room temperature). This indirect coating is superior to direct coating with the Pab240 antibody. The wells were then washed six times with an 25 automatic washer and used for the assay as follows: 50 microliters of cell lysate ~diluted lO-fold in the cell lysate diluent) and 100 microliters of mouse monoclonal anti-p53 antibody Pab240 ~diluted 20-fold in the PAb240 diluent~ were added and incubated for 3 h with shaking at 37 30 C ~air oven). After 6 washes, 100 microliter~well of serum sample ~diluted 10-fold in the serum sample diluent, in duplicate) and incubate for 1 h with shaking, at room temperature. After six washes, 100 microliters~well of an Alk=linP phosphatase-labeled goat anti-human i nglnhulin G-antibody ~diluted 15,000 fold in the GAHIg-ALP diluent) was added. The wells were incubated for 1 h with shaking at room temperature and washed six times. 100 microlitersiwell 2194~ 7 Wo9~/olgos ~CT~rS9 of the diluted FSAP substrate solution was added and incubated for 10 min with shaking at room temperature. 100 microliters/well of the developing solution, was added, mixed for 1 min and the fluorescence was measured on a Cyberfluor 615TM ~ nn~n~lyzer.
Each assay run was scrnmr~nied by a parallel run to assess any rnn~perific binding effects. This run was identical to the procedure described above but the cell lysate was replaced by the lysate diluent. Sera were considered positive for antihodies only if the signal with the lysate exceed the signal without the lysate by a factor oi 1.7 ~Hassapoqlidou 1992, supra).
T nAcsay Procedure Method B - ~icrotiter strips were coated as in Method A. Patient serum (200 microliters) was then incubated in tubes with 20 microliters of a 10-fold diluted c.ell lysate from Colo 3Z0 ~SR (+) cells, for 30 min at room temperature. The p53-supplemented sera (50 microliters, in duplicate) were then added to goat anti-mouse IgG-coated wells along with 100 microliters/well of mouse rlnn~l anti-p53 antibody Pab240, diluted 20 fold as in Method A. The wells were incubated at 37 C for 3 h with shaking and washed six times. 100 microliters/well of the rabbit anti-p53 polyclonal antibody (CM-1) diluted 5,000 fold in the C~-1 antibody diluent was added and 2S ir~lh~tPs for 1 h at room temperature with shaking. After washing six times lO0 microliters/well of the ~lk~l inP
phosphatase-labeled goat anti-rabbit ; Ir~lnhulin (GARIg-ALP) diluted 5Q00-fold in the GARIg-ALPP antibody diluent was added and incubated for 1 h at room temperature with shaking. The wells were washed six times and the procedure rnn~lnll~d as in Method A from the point o~ adding the FSAP substrate solution. Each serum sample was also assayed without the addition of the Colo 320 ~SR(+) cell lysate to assess the background signal. Sera were considered positive for antibodies only if the fluorescence signal in the presence of serum was less than 50~ of the 2 1 ~ 9 ~
~ wos6lol9li9 Fc~
fluorescence signal obtained with a 6% BSA solution as sample.
Quantification - Due to the lack of a suitable standard solution, an arbitrary system to calibrate Methods A and B was devised. Among the highly p53 antibody-positive - sera one was selected and arbitrarily defined to have a concentration of 20,480 Units~L. This serum sample as then used in dilutions to construct calibration curves for assays A and B from which the concentration of the other samples was calculated.
The results of the test identified the presence of anti-p53 antibodies in 15-16% of ovarian and colon cancer patients. Antibody prevalence was between 5-8% in patients with lung and breast tumors. There was a relatively low prevalence of detectable anti-p53 antibodies ~3-4%) in patients with pancreatic and prostate cancer and in patients with multiple myeloma or lymphoma. In patients with other ~-lign~nnie5 ~hepatoma, ~l~nn~, leukemia, Kaposi's sarcoma and testicular carcinoma~ the p53-antibody prevalence was similar to that of non-cancer patients ~less than 2%).
Patient reports were prepared for those samples which demonstrated positive results. All those test samples which were not positive for the p53 mutation in the protein i n~Csay were then analyzed at the next level in the hierarchy of the invention.
Level 2: DNA Fra t T~n~th~OII~rtitv An~lvs;q DNA is prepared from the patient qample using a Qiagen QIAamp Kit according to accompanying directions. Briefly, an aliquot of the blood sample, or a lymphocyte--containing iraction thereof, is cn-~;n~d with Proteinase K, mixed, and allowed to incubate to lyse the cells. Ethanol is added and the lysate is transferred to a QIAamp spin column from which DNA is ! ~vv~L~d after several washings.
Quantitative fragment length and amount analysis is performed to assay for l) the presence of insertion or deletion mutations: and 2) whether the patient is homozygous ~ ~ ~469~
WO 96/0190!1 PCT/U39~08605 or heterozygous for the insertion or deletion mutation.
To perform the analysis, the genomic D~A is amplified in three sets using multiplexing amplification primers. Eac.h 50 microliter multiplexed PCR reaction contains 0.5 mi~,o~L~rs genomic DNA, 150 ng or each primer, 3.6 mM each dNTP, 42.5 micrcgrams Bovine Serum Albumin, 5 units Taq polymerase in a buffer containing 10% DMSO, 16 mM [NH,)2So"
6.7 mM MgCl2, 6.8 micro Molar EDTA ~pH 8.0) and 1 mM ~-mercaptoethanol. The reaction mixture was initially incubated at 94 degrees C for 5 minutes and then subjected to 30 cycles of PCR in a Perkin-Elmer~Cetus thermocycler as follows:
Denaturation: 94 degrees C, 30 seconds Ann~linq 60, 62 or 64 degrees C ~pcn~ing on whether primer set A, B, or C is being amplified, respectively) for 50 secs.
Extension: 70 degrees C, 60 seconds; final extension at 72 degrees for 3 minutes The amplification of the eleven exons of the p53 gene 20 i5 advantageously carried out in three multiplex pools. In multiplex pool A, exons 1, 3, 4, 5, 6, 9, 10 and 11 are amplified lalong with a control sequence). The m~mbers of this pool are selected because they all use a hybridization temperature of 60~C, and none of the expected fragment 25 lengths will overlap in an electrophoresis gel. One of each pair of primers is labeled at the 5 prime end ~ith an idertifiable marker such as fluorescein, rhodamine or cyanine. The primers are:
P53-5XlMP
CGGATTACTT GCCCTTACTT GTCA [9EO l]
P53-3XlMP
CCCCAGCCCC AGCGATTTT [SEQ 2]
EXO~ 3 P53-5X3,4P
CATGGGACTG A~ ~s [SEQ 3]
0 9610190g P~u_,S,'( '~'-GGACGGCAAG GGGGACTGT [SEQ 4]
~ EXON 4 P53'5X4MP
GA~lG~ TTCA [SEQ 5]
P53-3X3,4P
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
E53-5X5,6P
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCC~ 'CCAG [SEQ 8]
~G~I~G ~r~Ar~Arr~ArA [SEQ 9]
P53-3X5,6P
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG ArArrAAGG [SEQ 11]
AArGGrA~TT TGAGTGTTAG A C [SEQ 12]
P53-5XlOP
TGATCCGTCA T~AAGTCAAA CAA [SEQ 13]
GT~rArGrAA GAATGTGGTT A [SEQ 14]
P53-5XllP
GGrArArArc CTCTCACTCA T [SEQ 15]
P53-3XllP
~ ~ 9~7 WO g6101~)g TGCTTCTGAC GCACACCTAT T [SEQ 16]
These primers result in amplified products with normal fragment lengths of 331bp for exon 1, 162 bp for exon 3, 382bp for exon 4, 268 bp for exon 5, 247bp for exon 6, 209bp for exon 9, 390bp for exon 10, and 256bp ~or exon 11. The control sequence produces a further fragment having a length which should not correspond to any of the expected lengths.
In multiplex pool B, exons 2 and 8 are amplified ~along with a control sequence~. The members of this pool are selected because they all use a hybridization temperature of 62~C, and none of the expected fragment lengths will overlap in an electrophoresis gel. One of each pair of primcrs is labeled at the 5 prime end with an identifiable marker such as fluorescein, rhodamine or cyanine. The primers are:
~Cr~GGTT GGAAÇCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19]
~A~.~.A~ GTGATAAAAG TÇAA [SE~ 20]
These primers result in amplified products with normal ~L , t lengths of 261bp for exon 2 and 320bp for exon 8.
The control sequence produces a further fragment having a length which should not correspond to any o~ the expected lengths.
Finally, in multiplex pool C, only exon 7 is amplified ~along with a control sequence). The primers for exon 7 require a hybridization temperature of 64~C unlike any of the other amplification primers. One of the pair of primers ~ w096~0lgo9 ~ 7 F~~
- 23 ~
is labeled at the 5 prime end with an identifiable marker such as fluorescein, rhn~Am;ne or cyanine. The primers are:
GGCGACAGAG CGAGATTCCA [SEQ 21]
GGGTCAGCGG CAA~.~b~b~.~ [SEQ 22]
These primers result in an amplified product with a normal fragment length of 286bp. l'he control sequence produces a further fragment havinq a length which should not correspond to this expected length.
After amplification, the products from each amplifica-tion reaction are denatured and loaded into a polyacrylamide gel for electrophoretic separation. In the preferred embod-iment, electrophoretic separation takes place in a semi-automated electrophoresis apparatus such as in a ph~r~ri~
A.L.F.~ automated sequencer. In another . '~ t, elec-trophoretic separation takes place in a microgel disclosed in US Patent Application 08~332,577, which is incorporated herein by reference. In either : ,o~li-- t, the amplifica-tion products migrate through the gel at a rate det~rminP~
by their length and are detected using the fluorescence of the fluorescent molecule ~either fluorescein, rhn~m;nr or cyanine) which was attached to the primers.
~he products of each amplification set are separated in a different lane of the gel, unless molecules which fluor-esce at different wavelengths have been used as labels on the primers, in which case the products may be run in the same lane, and distinguished by wavelength of fluorescence emission. The fragment sizes and amounts are compared to the expected sizes of the normal gene fragments. If length mutation is detected, then the sample is cnnr~ d to con-tain a mutation in the p53 gene. If the amount of amplified fragment is 25% or more below or above the amount of the wild type fragment ~using the amount of control fragment as a standard of comparison), then the sample is concluded to 21 94t~97 contain an LOH (loss of heteroz~gosity) or gene amplifica-tion mutation, respectively. A patient report was Drepared for those samples that are identified as having a fragment length or quantity mutation. Where no length or quantity mutation is detected, then the sample was re-PY~m;n~ using the next and final level of the hierarchy.
Level 3: DNA Seguence ~n~lvsis The group of patient samples that have proven negative for pS3 mutations under the protein i ~say and the fragment length/quantity analysis may be re-~Y~min~d for point mutations in the DNA sequence.
DNA from an individual patient is purified as in the fragment length/quantity analysis, above, Exon containing fragments of the p53 gene are amplified using primers and conditions listed in Table 1. The primers are the same as those used in the fragment length/quantity analysis.
However, in some cases the primers are used in different combinations. For example, because exons 5 and 6 lie in reasonably close proximity on genomic DNA, it is adequate for amplification to use the primer at the 5 prime end of exon 5 and at the 3 prime end of exon 6, and to amplify both exons together on a single fragment. The resulting amplified fragment is suitable for sequencing either exon 5 or exon 6. The same situation is found with exons 3 and 4.
TABLE 1 - PRE ~QU~N~1N~ AMPLIFICATION C~N~1'1'1UN~
EXON S' primer 3' primer initial denaturing anneal Exten~ion cyclec final denaturing temp/time temp/tim~ temp/ time exten~ion temp/time temp/time 1 pS3-SXlPCR pS3-3XlPCR 94'C/4 min 94-C/30 min 60~C/SO min 70'C/60 min 30 72'C/3 min 72'C/3 min 2 pS3-SX2PCR pS3-3X2PCR 94~C/4 min 94-C/30 min 62-C/SO min 7U'C/60 min 30 72-C/3 min 3 pS3-SX3PCR pS3-3X3PCR g4~C/4 min 94'C/30 min 60-C/SO min 70'C/60 min 30 72-C/3 min 4 pS3-SX4PCR pS3-3X4PCR 94-C/4 min 94nC/30 min 60'C/SO min 70-C/60 min 30 72~C/3 min S pS3-SXSPCR pS3-3XSPCR 94-C/4 min 94~C/30 min 60~C/SO min 70~C/60 min 3U 72~C/3 min 6 pS3-SX6PCR pS3-3X6PCR 94'C/4 min 94~C/30 min 60'C/SO min 70'C/60 min 30 72~C/3 min 7 pS3-SX7PCR pS3-3X7PCR 94-C/4 min 94'C/30 min 64"C/SO min 70'C/60 min 30 72'C/3 min O 8 pS3-SXaPCR pS3-3X8PCR 54-C/4 min s4-c/30 min 62'C/SO min 70~C/60 min 30 72-C/3 min g pS3-SX9PCR pS3-3X9PCR 94-C/4 min 94'C/30 min 60~C/SO min 70-C/60 min 30 72'C/3 min P53-SXlOPCR pS3-3XlOPCR 94-C/4 min 94'C/30 min 60'C/SO min 70'C/60 min 30 72-C/3 min 11 pS3-SXllPCR pS3-3XlOPCR 94'C/4 min 94~C/30 min 60~C/SO m~n 70'C/60 min 30 72~C/3 min 2 I q4G~7 WO96101909 F~
Once the sets of exons are amplified, DNA sequencing reactions may be performed on the amplified sample. Dideoxy sequencing primers have been developed for both strands of each exon ~except exon 3 which has only one se~uencing primer) of the p53 gene, and are listed below:
P53-5XlSEQ CGGATTACTT GCCCTTACTT GTCA~ [SEQ 11 P53-3XlSEQ crcrAr.cccc AGCGATTTT [SEQ 2]
P53-5X2SEQ CCAGGGTTGG AAGCGTCTC* [SEO 23]
P53-3X2SEQ GCTAGGGGGC l~GG~lIGG [SEQ 24]
P53-3X3SEQ ATGGGTGAAA AGAGCAGT~ [SEQ 251 P53-5X4SEQ GGGGCTGAGG ACCTGGTC [SEQ 26]
P53-3X4SEQ ATACGGCCAG GCATTGAA [SEQ 27]
P53-5X5SEQ CAe~ GCC CTGACTTT~ [SEQ 28]
P53-3X5SEQ CcTGGG~A~c CTGGGCAA [SEQ 29]
P53-5X6SEQ ~ GCCCA GGGTCCCC~ [SEQ 30]
P53-3X6SEQ CC~CTGACAA CCACCC [SEQ 31¦
P53-5X7SFQ ~lcCC~lGel TGCCACA~ [SEQ 32]
P53-3X7SEQ Tr~rrG~ GCAGAGG [SEQ 33]
P53-5X8SEQ ATGGGACAGG TAGGACC~ [SEQ 34l P53-3X8SEQ CATAACTGCA CCCTTGG [SEQ 35¦
2 ~ ~4697 WOg6/01909 r~:., P53-5X9SEQ GGAGGAGACC AAGGGTGC [SEQ 36]
P53-3X9SEQ GGAAACTTTC CACTTGA~ [SEQ 37]
P53-5XlOSEQ CCATCTTTTA ACTCAGGT~ [SEQ 38]
P53-3XlOSEQ CATGAAGGCA GGATGAG [SEQ 39]
P53-5XllSEQ AGACCCTCTC ACTCATG [SEQ 40]
P53-3XllSEQ CAAGCAAGGG TTCAAAG~ [SEQ 41]
The primers are generally nested inside the amplification primers, i.e. closer to the exon, although in some cases the preferred sequencing primer is in fact the amplification primer. The 5 prime sequencing primer provides the sequence from the sense strand; the 3 prime sequencing primer pro-vides the sequence from the anti-sense strand of the p53 gene. Only one of these primers needs to be used to obtain sequence from the exon in question. The preferred primer is marked with an asterisk in the list above. The preferred primer for sequencing is conjugated to a fluorescent mole-cule such as fluorescein, rhn~=min~ or cyanine for detec-tion, although other forms of detectable labels, including labeled nucleotides or dideoxynucleotides may be employed.
Dideoxy DNA sequencing is perfor~m~ed using the well known method of Sanger et al., NDNA sequencing with chain terminating inhibitors~, Proc Natl Ac~d Sci USA 74:5463-5467 (1977), as modified for use with Sequenase~ Version 2.0 (~nited States Biochemical Corporation, Cleveland OE) Prod-ucts of the DNA sequencing reaction are analyzed using a semi-automated electrophoresis apparatus as in the DNA
fragment lengthJquantity analysis described above.
Current epidemiological data was used to determine which gene fragments are preferably se~uenced first.
Mutations have been detected in all exons, but are extremely rare in exon 1 and in the 3'-end of exon 11. It is there-W096l0l909 21 qf~5~,~ r~.,.s fore preferable to sequence exons 2-10 ahead of exons 1 and 11. Currently the preferred order of seguencing begins with exon 6, then 7, then 5. The L . ; n i nq exons are sequenced in turn.
Samples wherein mutations are detected relative to the wild-type p53 gene are recorded and reported to the individual patient's file. h'here no mutation is identified, another exon con~f~in;nq fragment of the individual sample is sequenced. Again mutations are identified and reported.
If, after sequencing all the exon containing fragments of the gene, there are no mutations identified, it is concluded that the individual sample contains no pS3 mutation.
All final results of testing are reported to the patient file. The report is communicated to the patient by electronic tra~smission or written report, or both.
~Y;~MPT~T~'. 2 A second embodiment of the p53 assay skips the protein i o~say level and begins with the preparation of DNA
from a patient blood sample. Genomic DNA is pL~aLed fro,n a blood sample as in Example 1 and it is assayed according to the DNA measurement procedures of Example 1, including fragment length~guantity analysis and DNA sequence analysis. Patient reports are preparea when diagnosis of the presence or absence of p53 mutation is deten~ined.
Two general techniques of immunoassay have been employed. The first technique is an indirect method which detects anti-p53 antibodies that arise in some patients who have p53 mutations. Irnmunoassay tests for detecting anti-p53 antibodies in patient sera are currentl~ based on radio-active labeling, immunoprecipitation and immunoblotting.
All these methods are qualitative and time consuming and thus not suitable for screening larqe number of samples.
~Angelopoulou et al., ''Autoantibodies against the p53 tumor suppressor gene product quantified in cancer patient serum with time-resolved immunofluorometry~, Cancer J 6(6): 3i5-321 ~1993)).
The second technique of immunoassay directly detects mutant p53 protein. Such methods have been disclosed in at least two publications. sartek et al. disclosed an enzyme-linked immunosorbent assay for p53 which was applied for the measurernent of p53 in tumor tissue extracts (Oncogene, 6:
1699-1703 (l991)). ~assapoglidou et al developed a mono-clonal antibody useful for direct detection of mutant p53.
(Oncogene 8: 1501 1509 (1993).) DNA analysis of p53 rnutat:ions has been reported using various techniques Sinqle stranded conformational poly-morphism was used to detect mutant p53 by Ruypers et al.
("Detection of point mutations in DNA using capillary electrophoresis in a polymer network", J. Ckromat-ography 621:
14g-56 (1993j), Runnebaum et al. (NMutations in p53 as potential molecular markers for human breast cancer" Proc.
Nat~l Acad. Scl. USA 88: 10657-10661 ~1993) and by Felix et al ("Absence of hereditary p53 mutations in 10 familial AM~EN~ED SHEET
~ 1 94~
leukemia pedigrees", J Clin Invest 90: 653-8 (1992)). Actual genomic DNA sequencing diagno~,is was performed on relatively small groups of patients by Toguchida et al. ("Prevalence and spectrum of germline mutations of the pS3 gene among S patients with sarcoma'', N Engl J ~ed 326: 1301-8 (1992)) and by Malkin et al. ("Germline mutations of the pS3 tumor-suppressor gene in children and young adults with second malignant neoplasms', N En~l J ~.e~ 326: 1309-lS
(1992)). Detection of p53 mutations is also discussed in EP-A 0 390 323. The cDNA sequence of a larger group of patients (over 400) was reported in a Pharmacia LKs meeting publication (Andell et al. "A new approach in automated DNA
sequencing to analyse the pS3 gene in a large nu7nber of breast cancer patients", Pharmacia LKB meeting literature, lS 1994) These rapid DNA-based techniques have been used to detect mutations, but because they are so labor intensive, that large-scale screening tests are impractical (Harris et al., "Clinical implication.s of the pS3 tumor-suppressor gene', N En~1 J ~ed 329:1318-1327 (1993)).
Thus, the existing methods of diagnosis have been frustratingly unsatisfactory. Researchers have used either immunoassay or DNA analytical methods to diagnose pS3 mutation, even though such tests result in numerous false negatives. A method is required for rapid and cost effective diagnosis of p53 mutation in the millions of individuals who develop potentially lif.e threatening malignancies each year. Dunn et al., J. Ce71 sioche~ ~ol Suppl 18C: 199 (1994) briefly describes an assay system for Rsl mutations which is said to be cost effective, but does not describe such a system for pS3.
It is an object of this invention to provide a method for rapid and cost effective diagnosis of p53 mutations in a . sample of patients.
It is~a further object o~ this invention to provide DNA
se~uencing and amplification ~rimers specific for analysis of the pS3 gene from a patient sample.
AMENDE~ SHEET
2 1 9 4 ~I r 7f It is a further object of this invention to provide kits of DNA oligonucleotides for amplification and seq~encin~ of the p53 ~ene of a patient.
It is a further object o~ this in~ention to provide a method of generatin~ a pS3 mutation report which is used to AMENDED SIIEET
2 1 9'~ 7 provide appropriate genetic counseling to the patient and family upon whom the test i5 performed.
SUMM~RY OF T~ lNv~ s~ UN
Ir. accordance with the present invention, rapid and cost effective ~iA~nnsis of p53 mutations of a sample of patients is achieved by employing a selected plurality of diagnostic tools, in a hierarchy of increasing accuracy and cost per tool, in which each tool detects essentially no false positives. Diagnostic tests that may be included among the plurality of tests selected include, in order of increasing accuracy and cost:
~a) ; ~ACSays~ particularlY i naqsays for anti-p53 antibodies present in a patient sample;
lS ~b) analysis of DNA from a patient sample by quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene. Preferably, the amplification primers are mul~;~F~ so that more than one DNA fragment is amplified in a single vessel, using sets of primers which provide gene fragments of distinctive lengths when used to amplify a normal p53 gene; and ~c) analysis of DNA from a patiert sample by DNA
sequencing of the p53 gene beginning with the sequencing of those regions most likely to harbor point mutations, and proceeding to sequence regions less likely to harbor point mutations.
The present invention includes a multitude of amplifi-cation and sequencing primers. These primers, taken indivi-dually or as part of kits for the detection of p53 mutations represent a further aspect of the present invention.
Particularly preferred primers constitute sets that are compatible for coampli~ication and that produce amplified DNA fragments of distinctive lengths from other fragments amplified in the same set.
2~ ~fi~
. . ~ .
- 5 - .
test is performed. The generation of such reports, which may be in the form of a printed report, an electronic communication, such as a facsimile or electronic mail (e-mail~ transmission, or a posting of a data entry in a cornputer record relating to the patierlt~ is a further aspect ~ of the present invention.
PRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the hierarchy of p53 diagnosis as provided by the invention; and Fig. 2 outlines two methods of imrnunoassay for the presence of anti-p53 antibodies in patient sera.
DETAILED DESCRIPTI:ON OF THE INVE~TION
The present invention reLates to a method for the identification of p53 mutations, a method for generating reports for providing counsellng to patients and families of patients with p53 mutations, and to oligonucleotide primers and kits useful in practici~g these methods. The present invention utilizes the hierar:hical approach which is disclosed generally in US Patent Application Serial No.
08/271,946, and in a concurrently filed continuation-in-part thereof, PCT~US95~08606, (Attorney Docket No. VGEN.P-002-WO~
which is incorporated herein by reference.
The method for identification of p53 mutations in a sample of patients is based upon using diagnostic tools 1~
in a hierarchy of increasing accuracy and cost per tool; and 2~ in which each tool is of extremely high specificity and detects essentially no false positives. A sample which exhibits a positive result establishes the presence of a p53 mutation and a patient report can be prepared on this basis.
A sample which exhibits a negative result is thereafter --subjected to a more costly, but more accurate test to determine if a mutation is present.
Thus, as shown in Fig. 1, an example of such a hierarchy comprises, in order, an immunoassay step; a DNA
fragment length~quantity analysis; and DNA sequencing. In A~IIENDED SHEET
_ _ . _ _ _ . . .. ... . .. . .... .. . .
2 i ~4fiq7 W096/01909 r~.~.,.
~, Thus, as shown in Fig. 1, an example of such a hierarchy comprises, in order, an i oACSay step; a DNA
fragment length~quantity analysis; and DNA seguencing. In the exemplary results shown in Fig. 1, the first step has the advantage of low cost, but detects a positive result in only 15% of the patients. The second level of the test hierarchy is of moderate cost, but because of its increased accuracy it detects a positive result in 30~ (26 of 85) of the patients who tested negative using the ; --csay~
This means that the more expensive DNA Seguencing analysis need only be performed on 59% of the original patients.
Thus, the use of the hierarchical approach leads to a substantial reduction in the average cost of the test while providing high levels of accuracy for every patient, by having eliminated from the test pool those samples that are known to be positive.
TMMTlNf)~.~sAy pRo~n~
As illustrated in Pigure 1, the first level in the hierarchy of p53 diagnosis may be an ; -~csay. Because of the relatively low cost of i nAcsay~ it may be advantageously used to eliminate a significant number of patients from the sample pool prior to proceeding to the next level of the hierarchy. Methods of i nAqsay that can be employed at this first stage have been discovered by many researchers. The following list of papers all include methods for identifying p53 mutations by i -~Csay, though by no means is this list exclusive, and a practitioner skilled in the art may know of alternative methods of p53 ; nAcSay Angelopoulou et al., NAutoantibodies against the p53 tumor suppressor gene product ~uantified in cancer patient serum with time-resolved immunofluorometry~, Cancer ~ 6(6):
315-321 (19g3).
Bartek et al., Oncogene 6: 1699-1703 (1991~.
~ 1 q ~
~ W096lol909 ~ r~
_ - 7 -Crawford et al., "Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer~, Int J Cancer 30: 403-8 (1982).
~ Caron De Froentel et al., Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell lymphomaN, Int J Cancer 39: 185-9 ~1987).
Christopoulos et al., ~Oncogenes and tumor suppressor genes: new biochemical tests.~ CRC Cri t Rev Clin Lab Sci 29: 269-305 (1992).
Davidoff et al., UImmune response to p53 is fl~p~nflPnt upon p53 HSP70 complexes in breast cancers", Proc Natl Acad Sci USA 89: 3439-42 ~1992).
Hassapoglidou et al., ~Antibodies to the p53 tumor suppressor gene product quantified in cancer patient serum with a time-resolved immunofluorometric technique~, Clin Biochem 25:445-9 (1992).
Hassapoglidou et al., ~Quantification of p53 protein in tumor cell lines, breast tissue extracts and serum with time-resolved immunofluorometry , Oncogene 8:1501-1509 (1993).
~abrecque et al., ~Analysis of the anti-p53 antibody response in cancer patients~, Cancer Res 53: 3468-71 (1993).
Schlichtholz et al., ~he immune response to p53 in breast cancer patients is directed against i ofl~ n~nt epitopes unrelated to the mutational hot spot~, Cancer Res 52: 6380-84 (1992).
Volkmann et al., ~The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for ~-l;gn~ncy and in~epen~ent of the alpha-fetoprotein status~, Hepatology 18: 559-565 ~1993!
Winter et al., ~Development of antibodies against p53 in lung cancer patients appears to be dependent on the p53 mutation~, Cancer Res 52: 4168-74 (1992).
In selecting an ; o~Csay for use in the present method, it should be understood that some i ~cSayS only detect small subsets of p53 mutations, while other immuno-6 ~ ~
WOs6/0l90~ L _, ~,./{
.
assays can detect a wide variety of p53 mutations. In order to reduce the cost of diagnosis, it is advantageous to take into account the scope of detection of each ; ~s~y and select an i ~qsay which provides highly specific detec-tion for the greatest number of p53 mutations as theanalytical start point. If desired, a second ; ~csay may be used after the first immunoassay if it will identify other types of mutations not found by the first i ~.qsay.
This selection and sequential ordering of i ~qsays may continue until the patient pool is reduced to eliminate essentially all patients reasonably diagnosed with i ~qsay techniques before going on to other, more expensive and more accurate assays.
DNA ANATYSIS
The next level in the hierarchy of this invention test those patient samples which did not prove positi~e for p53 mutation based on i ~csay, be subjected to more accurate and more costly analysis. DNA analysis of patient samples is an non-limiting example of such an analysis. Within the general class of DNA analysis, however, there is a hierarchy of methods, each level in the hierarchy providing increasing accuracy but at increased cost. At the top end of this hierarchy (most accurate and expensive~is DNA sequencing.
DNA sequencing provides the maximum degree of accuracy because each base in DNA from the sample is i~n~if;e~ and compared to the wild-type sequence.
DNA analysis also provides the option of methods which are lower in the hierarchy than DNA sequencing but which offer greater accuracy than i ~SRy pLocedu-=s. A
non-limiting example of such a test is an assay for DNA
fragment length/quantity mutations. This fragment analysis can be used as an intermediate level in a three (or more) level hierarchy, or it can be used as the first level of a hierarchy in place of the immunoassay tests described above.
FRA~MT~T ANATYSIS
As illustrated in Figure 1, level two of the hierarchy of this invention may be DNA fragment length/ quantity ~ wog6/nl9U9 ~ I 9 ¢ 6 ~ 7 ~ s 5l analysis. In this test r one or more exons of the p53 gene are quantitatively amplified using primers designed to create amplification products of known length and the lengths and/or quantity of the amplified fragments are det~rm;n~d. If there is a variance between the length of any amplified exon, and the normal length of that exon, this is an indication of an insertion or deletion mutation in that exon. The quantity of amplified material from amplifi-cation of a sample exon may also reflect the loss of genetic material. In particular, by comparing the quantity of amplified materials produced to standards amplified from a null allele (0 copies of p53), a hemizygous standard ~l copy of p53), a wild type standard (2 copies of p53) and a trisomy standard (3 copies cf p53), the nature of the mutation may be further investigated.
The number of exons tested in step two of the hierarchy is a matter of choice for the user. For example, if after repeated testing on patient samples it is found that length or quantity mutations are rarely found in certain exons, it is preferable to test these exons last, after testing other exons to see if a mutation sufficient to cause the disease is detected, before incurring the expense to test these less likely exons. In testing these other exons, the user may choose to test them one at a time, or in one multiplexing group at a time. Alternatively, the user may choose to test all exons simultaneously at once.
When a length mutation is detected in this step of the hierarchy, it is not npcecs~ry to perform additional tests on the patient sample to complete the identification process. Preferably, however, the sequence of the mutated exon will be detPnm;n~d as part of the second level of the hierarchy to confirm that the mutation detected can in fact ~ be a cause of the observed disease.
Again, all those samples that prove positive for a ~ 35 length or quantity mutation are eliminated from the patient sample, and patient reports may be made for each patient eliminated. However, if no mutation is detected at this ~o s6/o]q()~ r~
level of the hierarchy, then a more accurate and costly analysis must be undertaken. DNA sequence analysis is a non-limiting example of such a level.
~he primers usea to amplify the sample DNA for fragment analysis are oligonucleotides of defined seguence selected to hybridize selectively with particular ;oortions of the p53 gene, generally introns. Each primer has bound to it a detectable label. A preferrea example of such a label is fluorescein, which is a standard label used in nucleic acid sequencing systems usinq laser light as a detection system.
Other detectable labels can also be employed, howeYer, in-cluding other fluorophores, radio-labels, ~h~ 1 couplers such as biotin which can be detected with streptavidin-linked enzymes, and epitope tags such as digoxigenin detec-ted using antibodies available from Boehringer-~r~he;m-While considerable variation is possible in these~uence of the primers used in amplifying the exons as part of the method of the present invention, the primers used in amplification and the conditions of the amplification are preferably optimized for use in the present invention.
Looking first at the primers used, it will be understood that in order to avoid the possibility of false positiYe results the primer pair, i.e., the combination of the 5'-primer and the 3~-primer for any given e~on must be unique 2S to the p53 so that only the p53 gene will be l;fied.
~his means that the primer sequences will be generally somewhat longer than the minimum which can be used as an amplification primer. Preferred primers are from 18 to 23 nucleotides in length, without intPrn~l homology or primer-primer homology. It is also desirable for the primers toform more stable duplexes with the target DNA at the primers~ 5'-ends than at their 3'-ends, because this leads to less false priming. Stability can be appro~imated by GC
content, since GC base pairs are more stable than A~ pairs, or by nearest neighbor thermodynamic parameters. Breslauer et al., "Predicting DNA duplex stability from base sequence", Proc. ~at'1 Acad. 5ci . USA 83~ 3746-3750 ~1986~.
WO 961019U~ 0' 5''~
' 11 -In addition, to ensure complete amplification of each exon, the two primers of a pair are preferably selected to hybridize in the introns immediately flanking the exon to be amplified using the primer pair.
Additional factors apply to the selection of primers ~ for multiplexed amplification of exons, i.e., where several exons are amplified concurrently in a single reaction mix-ture. These factors are discussed in Rylchik, W., Selection of Primers for Polymerase Chain Reaction", in Methods in Nolecular Biology, Vol. 1~: PCR Protocols: Current Nethods and Applications, White, B.A. ed., Humana Press, Totowa, N.J., 1993. Briefly, applying these factors, primer pairs are selected by position, similarity oE melting temperature, internal stability, absence of internal homology or homology to each other, i.e., they won't stick to each other or to themselves, and the 3'-end will not form a stable hairpin loop back on itself.
Thus, in the present case, the goal is to have sets of primer pairs with approximately the same thermal profile, so that they can be effectively coamplified together. This goal can be achieved by having groups of primer pairs with approximately the same lenqth and the same G~C content. In addition, it is preferred that the length of the gene region between the primer binding sites on a normal p53 gene differ for each exon to be multiplexed as a group. Differences of only one base in length are sufficient, provided a high resolution gel capable of resolving one base differences is used in analyzing the amplification products. However, greater differences in length are preferred.
To evaluate compatibility of primers for use in co; ,l;fication, it is desirable to determine the predicted melting temperature for each primer. This can be accom-plished in several ways. Por example, the melting temperature, Tm can be calculated using either of the following eguations:
Tm~~C) = 81.5 + 16.6 X log [Na] + 0.41 X (%GC)- 675/length 2 ~ q4 ~
WO~61019~)9 where [Na] is the concentration of sodium ions, and the ~GC
is in number percent, or T~ (~C) = 2 X (A + T) + 4 X (G + C) where A, ~, G, and C represent the number of adenosine, thy-midine, guanosine and cytosine residues in the primer. In general, primers for coamplification should be selected to have predicted melting temperatures differing by less than 4~C.
D~ SJ:~UII~:N"~ PiT,YSI~S
Figure 1 illustrates that the final element of the hierarchy of pS3 diagnosis is DNA sequencing. DNA sequence analysis involves detPrmin;n7 the sequence of the exons to locate the mutation.
Sequencing is expensive and so it may be desirable to use a sub-hierarchy within this level of testing to reduce the 1 ik~l ih~od af having to seguence all of the exons. In this case a suitable sub-hierarchy will be det~rm;n~d by identi~ying those exons wherein mutations are most likely to occur. Mutational hotspots have been identi$ied at codons 175, 245, 248, 249, 273 and 282, which correspond to exon 5 for the first listed hotspot, exon 6 for the second three listed hotspots and exon 7 for the latter two hotspots.
(Harris CC. p53: At the Crossroads of ~olecular Carcino-genesis and Rig~ Assessment. Science 1953; 262: 1980-1981.) In accordance with this sub-hierarchy, the first exons sequenced are those which are easy to seguence and which contain hotspots. Next, if no mutation is found, hotspot exons are se~uenced which are hard to seguence (i.e. are found empirically to give less clear results when treated similarly to other exonsl. Finally, the remaining exons are sequenced in descending order of the odds of findin~ a mutation based on prior epidemiological studies. (~he order of sequencing of the exons may change as patient data acr~ tes on the location of point mutation hotspo~s.l If 6 q '~
wos~/olsos r~ ;o~
no mutation has been detected after all the exons have been sequenced, then it is concluded that there is no mutation in the test sample and a report is generated accordingly.
A preferred method for DNA sequencing as part of the method of the invention involves amplifying the exon of interest and then detPrmiring its sequence. Amplification of each exon may be performed using the same primers employ ed for fragment length analysis, although the detectable label included for the fragment analysis is not nPrPcs~ry for amplification prior to sequencing. In this case, how-ever, multiplexed amplification cannot be used since only a single amplification product is desired.
The determination of the sequence of the amplified material may be carried out in any manner. A preferred approach, however, is the ~ell-known Sanger method involving a template-dependent primer extension reaction in the pres-ence of dideoxy chain terminating nucleotides. For this method, a sequencing primer is used which hybridizes to one chain of the amplified DNA. In general, sequencing primers are nested inside the amplification primers, although the amplification primers could be used for sequencing purposes if desired.
The amplification and sequencing primers used in the present invention are advantageously packaged as kits for the detection of mutations in the p53 gene. The primers may be packaged individually within the kit, or as mixtures of primers, sometimes referred to as Nprimer cocktails,N which are useful in a single reaction vessel. Such kits may contain a single pair of primers, useful for quantitative amplification of a single exon, or multiple pairs of primers useful for amplification of multiple exons. Such kits may further include amplification and/or sequencing primers for one or more exons. Such kits may also include reagents other than primers for use in the amplification reaction, such as a polymerase and buffers, but this is optional.
Preferred kits in accordance with the invention comprise a plurality of primer pairs useful in the , .. , ... . ... . ., .. . .... _ . _ . . . .. .. . _ 2 ~ ~697 ~O g61U1909 1 ~
co amplification of a plurality of exons of the p53 qene.
Primer pairs in such kits are selected to have a common melting temperature and to produce amplification products having differing lengths.
The followins non-limiting ~Amrl~c illustrate applications of the invention.
~x~MPL~ 1 In order to determine the presence or absence of p53 mutations in a sample of patients, the following hierarchy of tests was performed.
Level 1: I rAcSav: Detection of Anti-~53 Ant;ho~ies.
The two ~ethods of Angelopoulou and DiAmAn~is ~1993, supra) were employed to detect anti-p~3 antibodies in a patient sample. Figure 2 A illustrates the principle of Method A for anti-p53 antibody quantification in patient sera. In the presence of such antibodies, a ~sandwich" is formed between a mouse monoclonal anti-p53 antibody and human anti-pS3 antibodies, resulting in high fluorescence readings. Piqure 2 ~ illustrates the principle of ~ethod s for p53 antibody quant;ficati~n in patient sera. The left-hand panel shows that in the absence of anti-p53 antibodies in human serum, the added p53 antigen is measured as shown, giving rise to fluorescence. The right-hand panel shows that when anti-p53 Ant;ho~;~p are present in patient serum, they block the added p~3 antigen, resulting in ~ow fluorescence. No 5An~wi~b can be formed between the mou8e monoclo~al and rabbit polyclonal anti-p53 ~nt;ho~;~c. ~LP=
~1~A1 ;~P phosphatase; FSAP = fluorosalicyl phosphate; FSA =
fluorosalicylate; Ab=antibody.
Instrumentation and Materials - For measuring liquid-phase Tb3~ fluorescence in white microtiter wells, a Cyberfluor 615 I ~AnAly~er time resolved fluorometer was used as described elsewhere ~Christopoulous et al., supra;
Papana tasiou-Diamandi et al., ~Ultrasensitive thyrotropin ~ nn~cs~y based on en~ymatically amplified time-resolved 21 ~46~;~
~101909 .
fluorescence with a terbium chelateN, Clin Chem 38:545-48 ~1992)). The phosphate ester of 5-fluorosalicylic acid ~FSAP) was obtained from CyberFluor Inc., Toronto, Canada.
TbCl3. 6H20 was from GfS Chemicals, Columbus OH, USA. All other chemicals were form Sigma ~h~ Al Co., St. Louis, MO, - USA unless otherwise stated.
Solutions - The enzyme substrate buffer was a 0.1 mol/L
Tris solution, pH 9.1, containing 0.1 mol NaCl and 1 mmol MgCl2 per liter. The stock FSAP substrate solution was a 2 mol/L solution in 0.1 mol/L NaOX. Fresh FSAP substrate working solutions were prepared just before use by dilution ~10-fold) of the stock in the enzyme substrate buffer. The cell lysis buffer was a 20 mmol/L Tris solution, pH 8.1, containing 150 mmol NaCl, 10 g Nonidet P-40, 0.5 mmol phenylmethylsulfonyl fluoride, 2 mg leupeptin and 2 mg aprotinin per liter. The developing solution was a 1 mol/L
Tris base solution containing 0.4 mol NaOH, 3 mmol EDTA and 2 mmol TbCl3.6X2O per liter (r.o pH adjustment). The washing solution was a 5 mmol/L Tris buffer, pH 7.80, ront~;n;n~ 0.5 g Tween 20 and 150 mmol NaCl per liter. The coating antibody solution was a 50 mr,lol/L Tris buffer, pX 7.80, containing 0.5 g sodium azide per litcr. The CM-l antibody diluent was a 50 mmol/l Tris buffer, pH 7.80. containing 60g bovine serum albumin (BSA) per liter. The goat anti-rabbit immunoglobulin alkaline phosphatase conjugate (GARRIg-ALP~ diluent was a 50 mmol/l Tris buffer, pH 7.80, containing 60g BSA, 0.5 mol KCl and 100 ml goat serum per liter.
The cell lines used in this stud~ were colon carcinoma Colo 320 HSR(+) (MnrAkAm; et al., ~Detection of aberrations of the p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis", Cancer Res 51: 3356-61 (1991)); pancreatic car-cinoma MIA PaCa-2 (Barton et al. ~hn~rr~l; ties of the p53 tumor suppressor gene in hum~n pancreatic cancer~, Br J
Cancer 64:1076-82 1991)): breast carcinoma T-47D (Bartek et al., ~Genetic and immunochemical analysis of mutant p53 in WO96,~l90~
human breast cancer cell lines~, Oncoge~e 5:893-9 (1990));
and human erythroleukemia OCI M2 ~Singerland et al., ~Mutations of the p53 gene in human acute myelogenous leukemia~, Blood 77: 1503-7 (1991)).
These cell lines were cultured as described elsewhere;
they all have p53 gene mutations and overproduce mutant p53 protein (~cs~roglidou et al., 1993, supra). R~cn-~;n~nt wild type p53 protein, produced as described elscwhere (Wang et al., UThe murine p53 blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T
antigenn,Cell 57: 379-92 (1989)), was a gift by Dr. Carol Prives, Columbia University, New York.
Lysates from cell lines producing p53, or recombinant p53 were diluted in a 50 mmol/L Tris buffer, pH 7.80, containing 60 g of BSA per liter for Method A and in 100 goat serum for Method B. The mouse monnrlon~l anti-p53 capture antibody (Pab240) diluent was a 50 mmol/L Tris buffer, pH 7.80, containing 60g OSA and 0.5 mol KCl per liter. Serum samples were diluted in a serum diluent which is the same as the Pab240 diluent but supplemented with 10 normal goat serum and 2~ normal mouse serum. The goat anti-human immunoglobulin-alkaline phosphatase conjugate (GAHIg-AALP) diluent was the same as the GARIg-ALP diluent.
The mouse anti-p53 - ~l~n~l antibody Pab2A0 was produced as a tissue culture supernatant from a cell line donated to us by Dr. D.P. Lane, University of Dundee, U.K.
Its antibody concentration was approximately 30 micrograms/ml. The rabbit polyclonal anti-p53 antibody, CM-1, was obtained from Di ~io~ Labs, Nississauga Ontario.
The qoat anti-rabbit and goat anti-human antibodies, conjugated to ~lk~1 ;nP phosphatase, and the goat anti-mouse antibody, Fc fragment specific (GAMIg), all approximately 1 mg/ml, were obtained from Jackson Imunoresearch, West Grove, PA.
Patient Sera - Sera from cancer patients were stored at -70 C until analysis. Sera used were ~rom patients with breast (n=105), ovarian ~n=72~, colon (n=77) and pancreatic ~ WO9~0l909 1~
- 1'1 -cancer ~n=46). For correlation studies 38 p53 antibody-positive sera from patients with the above malirnAnripc was used plus sera from prostate, lymphoma, lung and multiple myeloma patients.
Procedures:
Cell Lysis - Cells from each cell line were grown until they reached approximately lOX6 cells/ml or 90~ confluency.
The cell pellet from a 15 ml culture was lysed in 300 microliters lysis buffer, for 30 min, on ice. The cell 10 extract was centrifuged at 12,000 X g for 10 min and the pellet discarded. The lysate was used within two hours.
Total protein was measured in the lysates with the birinrhnrinic acid ~BCA) assay, commercially available from Pierce r~Pm;rAl Co., Rockford IL. Lysates typically 15 contained 1-3 mg of protein per ml.
T~~lnn~,s~y Procedure, Method A - This method is a modification of an assay previously published ~HassApog'i~nu 1992, supra). White, opaque, 12-well microtiter strips ~from Dynatech laboratories, Alexandria, VA~ were coated 20 with goat anti-mouse immunoglobulin diluted 500-fold in the coating antibody diluent ~100 microliters~ 200 ng~well, overnight incubation at room temperature). This indirect coating is superior to direct coating with the Pab240 antibody. The wells were then washed six times with an 25 automatic washer and used for the assay as follows: 50 microliters of cell lysate ~diluted lO-fold in the cell lysate diluent) and 100 microliters of mouse monoclonal anti-p53 antibody Pab240 ~diluted 20-fold in the PAb240 diluent~ were added and incubated for 3 h with shaking at 37 30 C ~air oven). After 6 washes, 100 microliter~well of serum sample ~diluted 10-fold in the serum sample diluent, in duplicate) and incubate for 1 h with shaking, at room temperature. After six washes, 100 microliters~well of an Alk=linP phosphatase-labeled goat anti-human i nglnhulin G-antibody ~diluted 15,000 fold in the GAHIg-ALP diluent) was added. The wells were incubated for 1 h with shaking at room temperature and washed six times. 100 microlitersiwell 2194~ 7 Wo9~/olgos ~CT~rS9 of the diluted FSAP substrate solution was added and incubated for 10 min with shaking at room temperature. 100 microliters/well of the developing solution, was added, mixed for 1 min and the fluorescence was measured on a Cyberfluor 615TM ~ nn~n~lyzer.
Each assay run was scrnmr~nied by a parallel run to assess any rnn~perific binding effects. This run was identical to the procedure described above but the cell lysate was replaced by the lysate diluent. Sera were considered positive for antihodies only if the signal with the lysate exceed the signal without the lysate by a factor oi 1.7 ~Hassapoqlidou 1992, supra).
T nAcsay Procedure Method B - ~icrotiter strips were coated as in Method A. Patient serum (200 microliters) was then incubated in tubes with 20 microliters of a 10-fold diluted c.ell lysate from Colo 3Z0 ~SR (+) cells, for 30 min at room temperature. The p53-supplemented sera (50 microliters, in duplicate) were then added to goat anti-mouse IgG-coated wells along with 100 microliters/well of mouse rlnn~l anti-p53 antibody Pab240, diluted 20 fold as in Method A. The wells were incubated at 37 C for 3 h with shaking and washed six times. 100 microliters/well of the rabbit anti-p53 polyclonal antibody (CM-1) diluted 5,000 fold in the C~-1 antibody diluent was added and 2S ir~lh~tPs for 1 h at room temperature with shaking. After washing six times lO0 microliters/well of the ~lk~l inP
phosphatase-labeled goat anti-rabbit ; Ir~lnhulin (GARIg-ALP) diluted 5Q00-fold in the GARIg-ALPP antibody diluent was added and incubated for 1 h at room temperature with shaking. The wells were washed six times and the procedure rnn~lnll~d as in Method A from the point o~ adding the FSAP substrate solution. Each serum sample was also assayed without the addition of the Colo 320 ~SR(+) cell lysate to assess the background signal. Sera were considered positive for antibodies only if the fluorescence signal in the presence of serum was less than 50~ of the 2 1 ~ 9 ~
~ wos6lol9li9 Fc~
fluorescence signal obtained with a 6% BSA solution as sample.
Quantification - Due to the lack of a suitable standard solution, an arbitrary system to calibrate Methods A and B was devised. Among the highly p53 antibody-positive - sera one was selected and arbitrarily defined to have a concentration of 20,480 Units~L. This serum sample as then used in dilutions to construct calibration curves for assays A and B from which the concentration of the other samples was calculated.
The results of the test identified the presence of anti-p53 antibodies in 15-16% of ovarian and colon cancer patients. Antibody prevalence was between 5-8% in patients with lung and breast tumors. There was a relatively low prevalence of detectable anti-p53 antibodies ~3-4%) in patients with pancreatic and prostate cancer and in patients with multiple myeloma or lymphoma. In patients with other ~-lign~nnie5 ~hepatoma, ~l~nn~, leukemia, Kaposi's sarcoma and testicular carcinoma~ the p53-antibody prevalence was similar to that of non-cancer patients ~less than 2%).
Patient reports were prepared for those samples which demonstrated positive results. All those test samples which were not positive for the p53 mutation in the protein i n~Csay were then analyzed at the next level in the hierarchy of the invention.
Level 2: DNA Fra t T~n~th~OII~rtitv An~lvs;q DNA is prepared from the patient qample using a Qiagen QIAamp Kit according to accompanying directions. Briefly, an aliquot of the blood sample, or a lymphocyte--containing iraction thereof, is cn-~;n~d with Proteinase K, mixed, and allowed to incubate to lyse the cells. Ethanol is added and the lysate is transferred to a QIAamp spin column from which DNA is ! ~vv~L~d after several washings.
Quantitative fragment length and amount analysis is performed to assay for l) the presence of insertion or deletion mutations: and 2) whether the patient is homozygous ~ ~ ~469~
WO 96/0190!1 PCT/U39~08605 or heterozygous for the insertion or deletion mutation.
To perform the analysis, the genomic D~A is amplified in three sets using multiplexing amplification primers. Eac.h 50 microliter multiplexed PCR reaction contains 0.5 mi~,o~L~rs genomic DNA, 150 ng or each primer, 3.6 mM each dNTP, 42.5 micrcgrams Bovine Serum Albumin, 5 units Taq polymerase in a buffer containing 10% DMSO, 16 mM [NH,)2So"
6.7 mM MgCl2, 6.8 micro Molar EDTA ~pH 8.0) and 1 mM ~-mercaptoethanol. The reaction mixture was initially incubated at 94 degrees C for 5 minutes and then subjected to 30 cycles of PCR in a Perkin-Elmer~Cetus thermocycler as follows:
Denaturation: 94 degrees C, 30 seconds Ann~linq 60, 62 or 64 degrees C ~pcn~ing on whether primer set A, B, or C is being amplified, respectively) for 50 secs.
Extension: 70 degrees C, 60 seconds; final extension at 72 degrees for 3 minutes The amplification of the eleven exons of the p53 gene 20 i5 advantageously carried out in three multiplex pools. In multiplex pool A, exons 1, 3, 4, 5, 6, 9, 10 and 11 are amplified lalong with a control sequence). The m~mbers of this pool are selected because they all use a hybridization temperature of 60~C, and none of the expected fragment 25 lengths will overlap in an electrophoresis gel. One of each pair of primers is labeled at the 5 prime end ~ith an idertifiable marker such as fluorescein, rhodamine or cyanine. The primers are:
P53-5XlMP
CGGATTACTT GCCCTTACTT GTCA [9EO l]
P53-3XlMP
CCCCAGCCCC AGCGATTTT [SEQ 2]
EXO~ 3 P53-5X3,4P
CATGGGACTG A~ ~s [SEQ 3]
0 9610190g P~u_,S,'( '~'-GGACGGCAAG GGGGACTGT [SEQ 4]
~ EXON 4 P53'5X4MP
GA~lG~ TTCA [SEQ 5]
P53-3X3,4P
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
E53-5X5,6P
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCC~ 'CCAG [SEQ 8]
~G~I~G ~r~Ar~Arr~ArA [SEQ 9]
P53-3X5,6P
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG ArArrAAGG [SEQ 11]
AArGGrA~TT TGAGTGTTAG A C [SEQ 12]
P53-5XlOP
TGATCCGTCA T~AAGTCAAA CAA [SEQ 13]
GT~rArGrAA GAATGTGGTT A [SEQ 14]
P53-5XllP
GGrArArArc CTCTCACTCA T [SEQ 15]
P53-3XllP
~ ~ 9~7 WO g6101~)g TGCTTCTGAC GCACACCTAT T [SEQ 16]
These primers result in amplified products with normal fragment lengths of 331bp for exon 1, 162 bp for exon 3, 382bp for exon 4, 268 bp for exon 5, 247bp for exon 6, 209bp for exon 9, 390bp for exon 10, and 256bp ~or exon 11. The control sequence produces a further fragment having a length which should not correspond to any of the expected lengths.
In multiplex pool B, exons 2 and 8 are amplified ~along with a control sequence~. The members of this pool are selected because they all use a hybridization temperature of 62~C, and none of the expected fragment lengths will overlap in an electrophoresis gel. One of each pair of primcrs is labeled at the 5 prime end with an identifiable marker such as fluorescein, rhodamine or cyanine. The primers are:
~Cr~GGTT GGAAÇCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19]
~A~.~.A~ GTGATAAAAG TÇAA [SE~ 20]
These primers result in amplified products with normal ~L , t lengths of 261bp for exon 2 and 320bp for exon 8.
The control sequence produces a further fragment having a length which should not correspond to any o~ the expected lengths.
Finally, in multiplex pool C, only exon 7 is amplified ~along with a control sequence). The primers for exon 7 require a hybridization temperature of 64~C unlike any of the other amplification primers. One of the pair of primers ~ w096~0lgo9 ~ 7 F~~
- 23 ~
is labeled at the 5 prime end with an identifiable marker such as fluorescein, rhn~Am;ne or cyanine. The primers are:
GGCGACAGAG CGAGATTCCA [SEQ 21]
GGGTCAGCGG CAA~.~b~b~.~ [SEQ 22]
These primers result in an amplified product with a normal fragment length of 286bp. l'he control sequence produces a further fragment havinq a length which should not correspond to this expected length.
After amplification, the products from each amplifica-tion reaction are denatured and loaded into a polyacrylamide gel for electrophoretic separation. In the preferred embod-iment, electrophoretic separation takes place in a semi-automated electrophoresis apparatus such as in a ph~r~ri~
A.L.F.~ automated sequencer. In another . '~ t, elec-trophoretic separation takes place in a microgel disclosed in US Patent Application 08~332,577, which is incorporated herein by reference. In either : ,o~li-- t, the amplifica-tion products migrate through the gel at a rate det~rminP~
by their length and are detected using the fluorescence of the fluorescent molecule ~either fluorescein, rhn~m;nr or cyanine) which was attached to the primers.
~he products of each amplification set are separated in a different lane of the gel, unless molecules which fluor-esce at different wavelengths have been used as labels on the primers, in which case the products may be run in the same lane, and distinguished by wavelength of fluorescence emission. The fragment sizes and amounts are compared to the expected sizes of the normal gene fragments. If length mutation is detected, then the sample is cnnr~ d to con-tain a mutation in the p53 gene. If the amount of amplified fragment is 25% or more below or above the amount of the wild type fragment ~using the amount of control fragment as a standard of comparison), then the sample is concluded to 21 94t~97 contain an LOH (loss of heteroz~gosity) or gene amplifica-tion mutation, respectively. A patient report was Drepared for those samples that are identified as having a fragment length or quantity mutation. Where no length or quantity mutation is detected, then the sample was re-PY~m;n~ using the next and final level of the hierarchy.
Level 3: DNA Seguence ~n~lvsis The group of patient samples that have proven negative for pS3 mutations under the protein i ~say and the fragment length/quantity analysis may be re-~Y~min~d for point mutations in the DNA sequence.
DNA from an individual patient is purified as in the fragment length/quantity analysis, above, Exon containing fragments of the p53 gene are amplified using primers and conditions listed in Table 1. The primers are the same as those used in the fragment length/quantity analysis.
However, in some cases the primers are used in different combinations. For example, because exons 5 and 6 lie in reasonably close proximity on genomic DNA, it is adequate for amplification to use the primer at the 5 prime end of exon 5 and at the 3 prime end of exon 6, and to amplify both exons together on a single fragment. The resulting amplified fragment is suitable for sequencing either exon 5 or exon 6. The same situation is found with exons 3 and 4.
TABLE 1 - PRE ~QU~N~1N~ AMPLIFICATION C~N~1'1'1UN~
EXON S' primer 3' primer initial denaturing anneal Exten~ion cyclec final denaturing temp/time temp/tim~ temp/ time exten~ion temp/time temp/time 1 pS3-SXlPCR pS3-3XlPCR 94'C/4 min 94-C/30 min 60~C/SO min 70'C/60 min 30 72'C/3 min 72'C/3 min 2 pS3-SX2PCR pS3-3X2PCR 94~C/4 min 94-C/30 min 62-C/SO min 7U'C/60 min 30 72-C/3 min 3 pS3-SX3PCR pS3-3X3PCR g4~C/4 min 94'C/30 min 60-C/SO min 70'C/60 min 30 72-C/3 min 4 pS3-SX4PCR pS3-3X4PCR 94-C/4 min 94nC/30 min 60'C/SO min 70-C/60 min 30 72~C/3 min S pS3-SXSPCR pS3-3XSPCR 94-C/4 min 94~C/30 min 60~C/SO min 70~C/60 min 3U 72~C/3 min 6 pS3-SX6PCR pS3-3X6PCR 94'C/4 min 94~C/30 min 60'C/SO min 70'C/60 min 30 72~C/3 min 7 pS3-SX7PCR pS3-3X7PCR 94-C/4 min 94'C/30 min 64"C/SO min 70'C/60 min 30 72'C/3 min O 8 pS3-SXaPCR pS3-3X8PCR 54-C/4 min s4-c/30 min 62'C/SO min 70~C/60 min 30 72-C/3 min g pS3-SX9PCR pS3-3X9PCR 94-C/4 min 94'C/30 min 60~C/SO min 70-C/60 min 30 72'C/3 min P53-SXlOPCR pS3-3XlOPCR 94-C/4 min 94'C/30 min 60'C/SO min 70'C/60 min 30 72-C/3 min 11 pS3-SXllPCR pS3-3XlOPCR 94'C/4 min 94~C/30 min 60~C/SO m~n 70'C/60 min 30 72~C/3 min 2 I q4G~7 WO96101909 F~
Once the sets of exons are amplified, DNA sequencing reactions may be performed on the amplified sample. Dideoxy sequencing primers have been developed for both strands of each exon ~except exon 3 which has only one se~uencing primer) of the p53 gene, and are listed below:
P53-5XlSEQ CGGATTACTT GCCCTTACTT GTCA~ [SEQ 11 P53-3XlSEQ crcrAr.cccc AGCGATTTT [SEQ 2]
P53-5X2SEQ CCAGGGTTGG AAGCGTCTC* [SEO 23]
P53-3X2SEQ GCTAGGGGGC l~GG~lIGG [SEQ 24]
P53-3X3SEQ ATGGGTGAAA AGAGCAGT~ [SEQ 251 P53-5X4SEQ GGGGCTGAGG ACCTGGTC [SEQ 26]
P53-3X4SEQ ATACGGCCAG GCATTGAA [SEQ 27]
P53-5X5SEQ CAe~ GCC CTGACTTT~ [SEQ 28]
P53-3X5SEQ CcTGGG~A~c CTGGGCAA [SEQ 29]
P53-5X6SEQ ~ GCCCA GGGTCCCC~ [SEQ 30]
P53-3X6SEQ CC~CTGACAA CCACCC [SEQ 31¦
P53-5X7SFQ ~lcCC~lGel TGCCACA~ [SEQ 32]
P53-3X7SEQ Tr~rrG~ GCAGAGG [SEQ 33]
P53-5X8SEQ ATGGGACAGG TAGGACC~ [SEQ 34l P53-3X8SEQ CATAACTGCA CCCTTGG [SEQ 35¦
2 ~ ~4697 WOg6/01909 r~:., P53-5X9SEQ GGAGGAGACC AAGGGTGC [SEQ 36]
P53-3X9SEQ GGAAACTTTC CACTTGA~ [SEQ 37]
P53-5XlOSEQ CCATCTTTTA ACTCAGGT~ [SEQ 38]
P53-3XlOSEQ CATGAAGGCA GGATGAG [SEQ 39]
P53-5XllSEQ AGACCCTCTC ACTCATG [SEQ 40]
P53-3XllSEQ CAAGCAAGGG TTCAAAG~ [SEQ 41]
The primers are generally nested inside the amplification primers, i.e. closer to the exon, although in some cases the preferred sequencing primer is in fact the amplification primer. The 5 prime sequencing primer provides the sequence from the sense strand; the 3 prime sequencing primer pro-vides the sequence from the anti-sense strand of the p53 gene. Only one of these primers needs to be used to obtain sequence from the exon in question. The preferred primer is marked with an asterisk in the list above. The preferred primer for sequencing is conjugated to a fluorescent mole-cule such as fluorescein, rhn~=min~ or cyanine for detec-tion, although other forms of detectable labels, including labeled nucleotides or dideoxynucleotides may be employed.
Dideoxy DNA sequencing is perfor~m~ed using the well known method of Sanger et al., NDNA sequencing with chain terminating inhibitors~, Proc Natl Ac~d Sci USA 74:5463-5467 (1977), as modified for use with Sequenase~ Version 2.0 (~nited States Biochemical Corporation, Cleveland OE) Prod-ucts of the DNA sequencing reaction are analyzed using a semi-automated electrophoresis apparatus as in the DNA
fragment lengthJquantity analysis described above.
Current epidemiological data was used to determine which gene fragments are preferably se~uenced first.
Mutations have been detected in all exons, but are extremely rare in exon 1 and in the 3'-end of exon 11. It is there-W096l0l909 21 qf~5~,~ r~.,.s fore preferable to sequence exons 2-10 ahead of exons 1 and 11. Currently the preferred order of seguencing begins with exon 6, then 7, then 5. The L . ; n i nq exons are sequenced in turn.
Samples wherein mutations are detected relative to the wild-type p53 gene are recorded and reported to the individual patient's file. h'here no mutation is identified, another exon con~f~in;nq fragment of the individual sample is sequenced. Again mutations are identified and reported.
If, after sequencing all the exon containing fragments of the gene, there are no mutations identified, it is concluded that the individual sample contains no pS3 mutation.
All final results of testing are reported to the patient file. The report is communicated to the patient by electronic tra~smission or written report, or both.
~Y;~MPT~T~'. 2 A second embodiment of the p53 assay skips the protein i o~say level and begins with the preparation of DNA
from a patient blood sample. Genomic DNA is pL~aLed fro,n a blood sample as in Example 1 and it is assayed according to the DNA measurement procedures of Example 1, including fragment length~guantity analysis and DNA sequence analysis. Patient reports are preparea when diagnosis of the presence or absence of p53 mutation is deten~ined.
Claims (33)
1. A method for testing a patient sample for a disease-associated mutation in the p53 gene, comprising the steps of:
(a) selecting a hierarchy of assay techniques comprising at least a first and final assay, said first assay being selected to provide a highly specific test for the existence of the disease-associated mutation and said final assay being selected to provide a highly accurate and highly specific test for the existence of the disease associated mutation;
(b) analyzing the patient sample using the first assay; and, if the result of the first assay is negative for the presence of a disease-associated mutation, (c) analyzing the patient sample using the final assay.
(a) selecting a hierarchy of assay techniques comprising at least a first and final assay, said first assay being selected to provide a highly specific test for the existence of the disease-associated mutation and said final assay being selected to provide a highly accurate and highly specific test for the existence of the disease associated mutation;
(b) analyzing the patient sample using the first assay; and, if the result of the first assay is negative for the presence of a disease-associated mutation, (c) analyzing the patient sample using the final assay.
2. A method for testing a plurality of patients for a disease-associated mutation in the p53 gene comprising the steps of:
(a) performing an immunoassay on samples obtained from each of the plurality of patients, by combining a portion of sample with a specifically immunoreactive substance which forms an immunological reaction product by binding to p53 protein or anti-p53 antibodies and monitoring for formation of the immunological reaction product, (b) separating the samples into a first set which tested positive in the immunoassay and a second set which tested negative in the immunoassay; and (c) performing a DNA analysis having greater accuracy than the immunoassay on samples from the second set, but not on samples from the first set.
(a) performing an immunoassay on samples obtained from each of the plurality of patients, by combining a portion of sample with a specifically immunoreactive substance which forms an immunological reaction product by binding to p53 protein or anti-p53 antibodies and monitoring for formation of the immunological reaction product, (b) separating the samples into a first set which tested positive in the immunoassay and a second set which tested negative in the immunoassay; and (c) performing a DNA analysis having greater accuracy than the immunoassay on samples from the second set, but not on samples from the first set.
3. A method for testing a plurality of patients for a disease-associated mutation in the p53 gene comprising the steps of:
(a) amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon;
(b) separating the samples into a first set and a second set, wherein the first set contains samples where the length or quantity of the amplification fragments differed from the standard value and the second set contains samples where neither the length nor quantity differed from the standard value; and determining the sequence of DNA in at least one exon of the p53 gene on each patient sample in the second set, and comparing the sequence determined with the wild-type sequence of the p53 gene.
(a) amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon;
(b) separating the samples into a first set and a second set, wherein the first set contains samples where the length or quantity of the amplification fragments differed from the standard value and the second set contains samples where neither the length nor quantity differed from the standard value; and determining the sequence of DNA in at least one exon of the p53 gene on each patient sample in the second set, and comparing the sequence determined with the wild-type sequence of the p53 gene.
4. A method for generating a patient report on the presence or absence of p53 mutation comprising the steps of (a) obtaining a sample of patient tissue;
(b) performing an immunoassay on the sample by combining a portion of sample with a specifically immunoreactive substance which forms an immunological reaction product by binding to p53 protein or anti-p53 antibodies and monitoring for formation of the immunological reaction product, wherein the presence of absence of an immunological reaction product is indicative of the presence of a mutation;
(c) performing a DNA analysis having greater accuracy than the immunoassay on the sample if the no mutation was detected using the immunoassay; and (d) generating a report indicating whether a mutation was found in the sample.
(b) performing an immunoassay on the sample by combining a portion of sample with a specifically immunoreactive substance which forms an immunological reaction product by binding to p53 protein or anti-p53 antibodies and monitoring for formation of the immunological reaction product, wherein the presence of absence of an immunological reaction product is indicative of the presence of a mutation;
(c) performing a DNA analysis having greater accuracy than the immunoassay on the sample if the no mutation was detected using the immunoassay; and (d) generating a report indicating whether a mutation was found in the sample.
5. A method for generating a patient report on the presence or absence of p53 mutation comprising the steps of (a) obtaining a sample of patient tissue;
(b) amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon wherein a difference in the length or quantity of amplification fragments is indicative of a mutation;
(c) if no mutation was detected based on the length and quantity of amplification fragments, determining the sequence of DNA in at least one exon of the p53 gene on each patient sample in the second set, and comparing the sequence determined with the wild-type sequence of the p53 gene; and (d) generating a report indicating whether a mutation was found in the sample.
(b) amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon wherein a difference in the length or quantity of amplification fragments is indicative of a mutation;
(c) if no mutation was detected based on the length and quantity of amplification fragments, determining the sequence of DNA in at least one exon of the p53 gene on each patient sample in the second set, and comparing the sequence determined with the wild-type sequence of the p53 gene; and (d) generating a report indicating whether a mutation was found in the sample.
6. A method according to claim 1, wherein the first assay is immunoassay.
7. A method according to claims 1, 2 or 4, wherein the immunoassay detects the presence of anti-p53 antibodies.
8. A method according to claim 1, 6 or 7, wherein the final assay is a DNA analysis.
9. A method according to claims 2, 4, or 8, wherein the DNA analysis performed comprises the steps of determining the sequence of at least one exon of the p53 gene and comparing the sequence determined to the wild-type gene.
10. A method according to claims 3, 5 or 9, wherein the exons are analyzed by sequence analysis in an order determined by the frequency of disease-associated point mutations within the exons, and wherein the sequences of exons having a lower frequency of point mutations are determined only if no mutation is detected by the sequencing of higher frequency exons.
11. A method according to claim 3, 5, 9 or 10, wherein at least one exon is sequenced using a sequencing primer selected from among:
CGGATTACTT GCCCTTACTT GTCA [SEQ 1];
CCCCAGCCCC AGCGATTTT [SEQ 2];
CCAGGGTTGG AAGCGTCTC [SEQ 23];
GCTAGGGGGC TGGGGTTGG [SEQ 24];
ATGGGTGAAA AGAGCAGT [SEQ 25];
GGGGCTGAGG ACCTGGTC [SEQ 26];
ATACGGCCAG GCATTGAA [SEQ 27];
CACTTGTGCC CTGACTTT [SEQ 28];
CCTGGGGACC CTGGGCAA [SEQ 29];
TGGTTGCCCA GGGTCCCC [SEQ 30];
CCACTGACAA CCACCC [SEQ 31];
CTCCCCTGCT TGCCACA [SEQ 32];
TCAGCGGCAA GCAGAGG [SEQ 33];
ATGGGACAGG TAGGACC [SEQ 34];
CATAACTGCA CCCTTGG [SEQ 35];
GGAGGAGACC AAGGGTGC [SEQ 36];
GGAAACTTTC CACTTGA [SEQ 37];
CCATCTTTTA ACTCAGGT [SEQ 38];
CATGAAGGCA GGATGAG [SEQ 39];
AGACCCTCTC ACTCATG [SEQ 40]; and CAAGCAAGGG TTCAAAG [SEQ 41].
CGGATTACTT GCCCTTACTT GTCA [SEQ 1];
CCCCAGCCCC AGCGATTTT [SEQ 2];
CCAGGGTTGG AAGCGTCTC [SEQ 23];
GCTAGGGGGC TGGGGTTGG [SEQ 24];
ATGGGTGAAA AGAGCAGT [SEQ 25];
GGGGCTGAGG ACCTGGTC [SEQ 26];
ATACGGCCAG GCATTGAA [SEQ 27];
CACTTGTGCC CTGACTTT [SEQ 28];
CCTGGGGACC CTGGGCAA [SEQ 29];
TGGTTGCCCA GGGTCCCC [SEQ 30];
CCACTGACAA CCACCC [SEQ 31];
CTCCCCTGCT TGCCACA [SEQ 32];
TCAGCGGCAA GCAGAGG [SEQ 33];
ATGGGACAGG TAGGACC [SEQ 34];
CATAACTGCA CCCTTGG [SEQ 35];
GGAGGAGACC AAGGGTGC [SEQ 36];
GGAAACTTTC CACTTGA [SEQ 37];
CCATCTTTTA ACTCAGGT [SEQ 38];
CATGAAGGCA GGATGAG [SEQ 39];
AGACCCTCTC ACTCATG [SEQ 40]; and CAAGCAAGGG TTCAAAG [SEQ 41].
12. A method according to claim 1, wherein the first assay is a quantitative amplification of at least one p53 exon using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene.
13. A method according to claim 1, wherein the hierarchy selected further comprises an intermediate assay effective to detect at least some mutations which were not detected using the first assay, but which is not as accurate as the final assay.
14. A method according to claim 13, wherein the intermediate assay is a quantitative amplification of p53 exons using amplification primers complementary to intron regions flanking each exon and examination of the length or quantity of each amplified fragment for nucleotide insertions or deletions relative to the normal p53 gene.
15. A method according to claims 2, 4, or 8, wherein the DNA analysis performed comprises the steps of:
amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon, wherein a difference in the length or quantity of the amplification fragments is indicative of the presence of a mutation; and if no mutation was detected based on the length and quantity of the amplification fragment, determining the sequence of DNA in at least one exon of the p53 gene and comparing the seguence determined with the wild-type sequence of the p53 gene.
amplifying at least one exon of the p53 gene to produce amplification fragments and comparing the length and quantity of the amplification fragments to standard values for the amplified exon, wherein a difference in the length or quantity of the amplification fragments is indicative of the presence of a mutation; and if no mutation was detected based on the length and quantity of the amplification fragment, determining the sequence of DNA in at least one exon of the p53 gene and comparing the seguence determined with the wild-type sequence of the p53 gene.
16. A method according to claims 3, 5, 12, 14 or 15, wherein the at least some of the exons are coamplified in a single amplification reaction mixture.
17. A method according to claim 16, wherein exons 1, 3, 4, 5, 6, 9 and 10 of the p53 gene are coamplified.
18. A method according to claim 17, wherein the exons are coamplified using primers of the sequence:
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CGGATTACTT AGCGATTTT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 16].
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CGGATTACTT AGCGATTTT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 16].
19. A method according to claim 16, wherein exons 2 and 8 of the p53 gene are coamplified.
20. A method according to claim 19, wherein the exons are coamplified using primers of the sequence:
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
21. A kit for the identification of mutations in the p53 gene, comprising at least one primer pair for amplification of an exon of the p53 gene, each member of said primer pair being labeled with a detectable label.
22. A kit according to claim 21, wherein the kit comprises a primer pair selected from the group consisting of:
(a) CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
and CCCCAGCCCC AGCGATTTT [SEQ 2];
(b) CATGGGACTG ACTTTCTGCT [SEQ 3]
and GGACGGCAAG GGGGACTGT [SEQ 4];
(c) CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
and AAAGAAATGC AGGGGGATAC GG [SEQ 6];
(d) TGTTCACTTG TGCCCTGACT [SEQ 7]
and CAGCCCTGTC GTCTCTCCAG [SEQ 8];
(e) CTGGGGCTGG AGAGACGACA [SEQ 9]
and GGAGGGCCAC TGACAACCA [SEQ 10];
(f) GCGGTGGAGG AGACCAAGG [SEQ 11]
and AACGGCATTT TGAGTGTTAG A C [SEQ 12];
(g) TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
and GTGGAGGCAA GAATGTGGTT A [SEQ 14];
(h) GGCACAGACC CTCTCACTCA T [SEQ 15]
and TGCTTCTGAC GCACCCTAT T [SEQ 16];
(i) ACCCAGGGTT GGAAGCGTCT [SEQ 17]
and GACAAGAGCA GAAAGTCAGT CC [SEQ 18];
(j) GACAAGGGTG GTTGGGAGTA GATG [SEQ 19]
and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20]; and (k) GGCGACAGAG CGAGATTCCA [SEQ 21]
and GGGTCAGCGG CAAGCAGAGG [SEQ 22].
(a) CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
and CCCCAGCCCC AGCGATTTT [SEQ 2];
(b) CATGGGACTG ACTTTCTGCT [SEQ 3]
and GGACGGCAAG GGGGACTGT [SEQ 4];
(c) CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
and AAAGAAATGC AGGGGGATAC GG [SEQ 6];
(d) TGTTCACTTG TGCCCTGACT [SEQ 7]
and CAGCCCTGTC GTCTCTCCAG [SEQ 8];
(e) CTGGGGCTGG AGAGACGACA [SEQ 9]
and GGAGGGCCAC TGACAACCA [SEQ 10];
(f) GCGGTGGAGG AGACCAAGG [SEQ 11]
and AACGGCATTT TGAGTGTTAG A C [SEQ 12];
(g) TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
and GTGGAGGCAA GAATGTGGTT A [SEQ 14];
(h) GGCACAGACC CTCTCACTCA T [SEQ 15]
and TGCTTCTGAC GCACCCTAT T [SEQ 16];
(i) ACCCAGGGTT GGAAGCGTCT [SEQ 17]
and GACAAGAGCA GAAAGTCAGT CC [SEQ 18];
(j) GACAAGGGTG GTTGGGAGTA GATG [SEQ 19]
and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20]; and (k) GGCGACAGAG CGAGATTCCA [SEQ 21]
and GGGTCAGCGG CAAGCAGAGG [SEQ 22].
23. A kit according to claim 21 or 22, further comprising at least one p53 sequencing primer for the exon.
24. A kit according to claim 23, wherein the sequencing primer is selected from the group consisting of CGGATTACTT GCCCTTACTT GTCA [SEQ 1], CCCCAGCCCC AGCGATTTT [SEQ 2], CCAGGGTTGG AAGCGTCTC [SEQ 23], GCTAGGGGGC TGGGGTTGG [SEQ 24], ATGGGTGAAA AGAGCAGT [SEQ 25], GGGGCTGAGG ACCTGGTC [SEQ 26], ATACGGCCAG GCATTGAA [SEQ 27], CACTTGTGCC CTGACTTT [SEQ 28], CCTGGGGACC CTGGGCAA [SEQ 29], TGTGTGCCCA GGGTCCCC [SEQ 30], CCACTGACAA CCACCC [SEQ 31], CTCCCCTGCT TGCCACA [SEQ 32], TCAGCGGCAA GCAGAGG [SEQ 33], ATGGGACAGG TAGGACC [SEQ 34], CATAACTGCA CCCTTGG [SEQ 35], GGAGGAGACC AAGGGTGC [SEQ 36], GGAAACTTTC CACTTGA [SEQ 37], CCATCTTTTA ACTCAGGT [SEQ 38], CATGAAGGCA GGATGAG [SEQ 39], AGACCCTCTC ACTCATG [SEQ 40], and CAAGCAAGGG TTCAAAG [SEQ 41].
25. A kit according to claims 21-24, wherein the kit comprises at least one primer cocktail containing a mixture of primers effective to coamplify a plurality of exons of the p53 gene.
26. A kit according to claim 25, wherein the primer cocktail contains a mixture of primers effective to amplify exons 2 and 8 of the p53 gene.
27. A kit according to claim 26, wherein the primer cocktail comprises primers of the sequence:
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
28. A kit according to claim 25, wherein the primer cocktail contains a mixture of primers effective to amplify exons 1, 3, 4, 5, 6, 9 and 10 of the p53 gene.
29. A kit according to claim 28, wherein the primer cocktail comprises primers of the sequence:
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CCCCAGCCCC AGCGATITT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 161.
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CCCCAGCCCC AGCGATITT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 161.
30. A primer cocktail comprising a plurality of oligonucleotide primers for amplification of exons 2 and 8 of the p53 gene.
31. A primer cocktail according to claim 30, wherein the primer cocktail comprises primers of the sequence:
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
ACCCAGGGTT GGAAGCGTCT [SEQ 17]
GACAAGAGCA GAAAGTCAGT CC [SEQ 18]
GACAAGGGTG GTTGGGAGTA GATG [SEQ 19] and GCAAGGAAAG GTGATAAAAG TGAA [SEQ 20].
32. A primer cocktail comprising a plurality of oligonucleotide primers for amplification of exons 1, 3, 4 5, 6, 9 and 10 of the p53 gene.
33. A primer cocktail according to claim 32, wherein the primer cocktail comprises primers of the sequence:
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CCCCAGCCCC AGCGATTTT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 16].
CGGATTACTT GCCCTTACTT GTCA [SEQ 1]
CCCCAGCCCC AGCGATTTT [SEQ 2]
CATGGGACTG ACTTTCTGCT [SEQ 3]
GGACGGCAAG GGGGACTGT [SEQ 4]
CTGGTCCTCT GACTGCTCTT TTCA [SEQ 5]
AAAGAAATGC AGGGGGATAC GG [SEQ 6]
TGTTCACTTG TGCCCTGACT [SEQ 7]
CAGCCCTGTC GTCTCTCCAG [SEQ 8]
CTGGGGCTGG AGAGACGACA [SEQ 9]
GGAGGGCCAC TGACAACCA [SEQ 10]
GCGGTGGAGG AGACCAAGG [SEQ 11]
AACGGCATTT TGAGTGTTAG AC [SEQ 12]
TGATCCGTCA TAAAGTCAAA CAA [SEQ 13]
GTGGAGGCAA GAATGTGGTT A [SEQ 14]
GGCACAGACC CTCTCACTCA T [SEQ 15] and TGCTTCTGAC GCACACCTAT T [SEQ 16].
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/271,946 US5545527A (en) | 1994-07-08 | 1994-07-08 | Method for testing for mutations in DNA from a patient sample |
US08/271,946 | 1994-07-08 | ||
US08/388,381 US5552283A (en) | 1994-07-08 | 1995-02-14 | Method, reagents and kit for diagnosis and targeted screening for P53 mutations |
US08/388,381 | 1995-02-14 |
Publications (1)
Publication Number | Publication Date |
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CA2194697A1 true CA2194697A1 (en) | 1996-01-25 |
Family
ID=26955205
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002194697A Abandoned CA2194697A1 (en) | 1994-07-08 | 1995-07-07 | Method, reagents and kit for diagnosis and targeted screening for p53 mutations |
Country Status (6)
Country | Link |
---|---|
US (1) | US6071726A (en) |
EP (1) | EP0770145B1 (en) |
AU (1) | AU3094895A (en) |
CA (1) | CA2194697A1 (en) |
DE (1) | DE69528703T2 (en) |
WO (1) | WO1996001909A1 (en) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6403303B1 (en) * | 1996-05-14 | 2002-06-11 | Visible Genetics Inc. | Method and reagents for testing for mutations in the BRCA1 gene |
US6013436A (en) * | 1994-07-08 | 2000-01-11 | Visible Genetics, Inc. | Compositions and methods for diagnosis of mutation in the von Hippel-Lindau tumor suppressor gene |
US5830657A (en) * | 1996-05-01 | 1998-11-03 | Visible Genetics Inc. | Method for single-tube sequencing of nucleic acid polymers |
US5888736A (en) * | 1995-12-22 | 1999-03-30 | Visible Genetics, Inc. | Method, compositions and kit for detection and identification of microorganisms |
US5789168A (en) * | 1996-05-01 | 1998-08-04 | Visible Genetics Inc. | Method for amplification and sequencing of nucleic acid polymers |
US6413718B1 (en) | 1996-05-01 | 2002-07-02 | Visible Genetics Inc. | Method for sequencing of nucleic acid polymers |
US6083699A (en) * | 1996-05-01 | 2000-07-04 | Visible Genetics Inc. | Method for bi-directional sequencing of nucleic acid polymers |
EP0914468B1 (en) * | 1996-05-01 | 2002-08-28 | Visible Genetics Inc. | Method for sequencing of nucleic acid polymers |
US6214555B1 (en) | 1996-05-01 | 2001-04-10 | Visible Genetics Inc. | Method compositions and kit for detection |
US5830665A (en) * | 1997-03-03 | 1998-11-03 | Exact Laboratories, Inc. | Contiguous genomic sequence scanning |
US6333179B1 (en) | 1997-06-20 | 2001-12-25 | Affymetrix, Inc. | Methods and compositions for multiplex amplification of nucleic acids |
EP1017856A1 (en) * | 1997-09-26 | 2000-07-12 | Visible Genetics Inc. | Method and kit for evaluation of hiv mutations |
AU2001249386A1 (en) * | 2000-03-22 | 2001-10-03 | Quantum Dot Corporation | Methods of using semiconductor nanocrystals in bead-based nucleic acid assays |
US20030049635A1 (en) * | 2000-11-08 | 2003-03-13 | City Of Hope | Measurement of mutation load using the p53 gene in human cells from paraffin embedded tissues |
EP2474624B1 (en) | 2011-01-05 | 2016-08-17 | Daniela Kandioler | Response prediction in cancer treatment (p53 adapted cancer therapy) |
EP2474625B1 (en) | 2011-01-05 | 2016-11-02 | Daniela Kandioler | Method for determining the p53 status of a tumour |
US9493813B2 (en) * | 2011-02-14 | 2016-11-15 | Beth Israel Deaconess Medical Center, Inc. | Modulation of phosphatidylinositol-5-phosphate-4-kinase activity |
Family Cites Families (5)
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DE69032864T3 (en) * | 1989-03-29 | 2013-01-31 | The Johns Hopkins University | Evidence of failure of the wild-type p53 gene |
WO1994000603A1 (en) * | 1992-06-26 | 1994-01-06 | The Trustees Of Princeton University | Method for detecting pre-cancerous or cancerous cells using p90 antibodies or probes |
WO1992000311A1 (en) * | 1990-06-27 | 1992-01-09 | Princeton University | Probes for detecting mutant p53 |
US5403707A (en) * | 1993-05-14 | 1995-04-04 | Eastman Kodak Company | Diagnostic compositions, elements, methods and test kits for amplification and detection of retroviral DNA using primers having matched melting temperatures |
US5545527A (en) * | 1994-07-08 | 1996-08-13 | Visible Genetics Inc. | Method for testing for mutations in DNA from a patient sample |
-
1995
- 1995-07-07 EP EP95926644A patent/EP0770145B1/en not_active Expired - Lifetime
- 1995-07-07 CA CA002194697A patent/CA2194697A1/en not_active Abandoned
- 1995-07-07 US US08/765,626 patent/US6071726A/en not_active Expired - Lifetime
- 1995-07-07 AU AU30948/95A patent/AU3094895A/en not_active Abandoned
- 1995-07-07 DE DE69528703T patent/DE69528703T2/en not_active Expired - Lifetime
- 1995-07-07 WO PCT/US1995/008605 patent/WO1996001909A1/en active IP Right Grant
Also Published As
Publication number | Publication date |
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US6071726A (en) | 2000-06-06 |
AU3094895A (en) | 1996-02-09 |
EP0770145A1 (en) | 1997-05-02 |
WO1996001909A1 (en) | 1996-01-25 |
DE69528703T2 (en) | 2003-07-17 |
DE69528703D1 (en) | 2002-12-05 |
EP0770145B1 (en) | 2002-10-30 |
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