Methods for the detection of loss of heterozygosity

• US 5,928,870 A
• Filed: 06/16/1997
• Issued: 07/27/1999
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Exact Laboratories, Inc.
• Inventors: Shuber, Anthony P., Lapidus, Stanley N.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/876,857
• Time in Patent Office: 771 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels

Abstract

Methods are provided for detecting loss of heterozygosity in a nucleic acid sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.

Claims

1. A method for detecting the presence of a mutant nucleic acid in a sample population, comprising the steps of:

a) introducing a first radionucleotide to a sample population suspected to contain a subpopulation of a nucleic acid mutant, wherein said first radionucleotide hybridizes to a first wild-type nucleic acid target, a subpopulation of which suspected to be mutated in the sample;

b) introducing a second radionucleotide to the sample, wherein said second radionucleotide hybridizes to a second wild-type nucleic acid target in the sample

c) washing said sample to remove unhybridized first and second radionucleotides;

d) determining a number X of radioactive decay events associated with said first radionucleotide;

e) determining a number Y of radioactive decay events associated with said second radionucleotide;

f) determining whether a difference exists between number X and number Y, the presence of a statistically-significant difference being indicative of the presence of a mutation in said sample.

2. The method of claim 1 wherein said first radionucleotide is capable of hybridizing to a nucleic acid in the sample that is suspected to be mutated in cancer or precancer;

and said second radionucleotide is capable of hybridizing to a nucleic acid in the sample that is not mutated in cancer or precancer.

3. The method of claim 1 wherein said first radionucleotide is capable of hybridizing to a portion of the maternal allele at a genetic locus;

and said second radionucleotide is capable of hybridizing to a portion of the paternal allele at said locus.

4. The method of claim 1 further comprising the step of isolating said first radionucleotide specifically bound to a first target nucleic acid, and said second radionucleotide specifically bound to a second target nucleic acid.

5. The method of claim 4 wherein said isolating step is selected from the group consisting of gel electrophoresis, chromatography, and mass spectrometry.

6. The method of claim 4 wherein said number X is correlated with a number X1 of molecules of said first nucleic acid, and said number Y is correlated with a number Y1 of molecules of said second nucleic acid.

7. The method of claim 1 wherein at least one of said first and second radionucleotides is a chain terminator nucleotide.

8. The method of claim 1 wherein at least one of said first and second radionucleotides is an oligonucleotide.

9. The method of claim 1 wherein said radionucleotides are labeled with an isotope selected from the group consisting of 32P, 33P, 35S, 125I and 14C.

10. The method of claim 1 wherein each of said first and second radionucleotides are labeled with a different isotope.

11. The method of claim 10 wherein said numbers X and Y are determined by coincidence counting.

20. The method of claim 1, wherein said sample comprises cellular material from a population of patients.

21. The method of claim 20, wherein said population of patients is healthy.

22. The method of claim 20, wherein said population of patients has a disease suspected to be associated with said mutant nucleic acid.

23. The method of claim 20, wherein said disease is cancer.

24. The method of claim 1, wherein said mutant nucleic acid is an allelic variant.

25. The method of claim 24, wherein said variant is a single nucleotide polymorphism.

12. A method for determining the number of molecules of a nucleic acid comprising the steps of:

a) exposing a sample to a plurality of first radionucleotides;

b) isolating radionucleotides specifically bound to first target nucleic acid molecules;

c) determining a number of radioactive decay events associated with the radionucleotides of step b);

d) calculating a number of molecules of said sequence as equivalent to said number of radioactive decay events.

13. A method for detecting the presence of a mutation in a nucleic acid, comprising the steps of:

a) exposing a sample to a plurality of a oligonucleotide;

b) performing a primer extension reaction in the presence of a plurality of a chain terminating nucleotide, to generate extension products of said oligonucleotide;

c) determining the size of the extension products, the presence of extension products of different sizes being indicative of the presence of a mutation.

14. The method of claim 13 wherein said oligonucleotide is capable of hybridizing to a member selected from the group consisting of a maternal allele and a paternal allele of the same genetic locus.

15. The method of claim 13 wherein said oligonucleotide is labeled.

16. The method of claim 13 wherein said terminating nucleotide is labeled.

17. The method of claims 15 or 16 wherein said label is a radioactive isotope.

18. The method of claim 13 wherein said extension reaction is performed in the presence of at least two differentially labeled chain terminating nucleotides.

19. A method for detecting loss of heterozygosity in a nucleic acid, comprising the steps of:

a) contacting a sample with a radionucleotide;

b) isolating a nucleic acid specifically bound to said radionucleotide;

c) determining a number of radioactive decay events associated with said nucleic acid;

d) comparing said number to a reference number,

wherein a statistically significant difference between said number and said reference number is indicative of loss of heterozygosity.