Primer extension methods for detecting nucleic acids

• US 6,566,101 B1
• Filed: 04/27/1998
• Issued: 05/20/2003
• 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 Sciences Corporation
• Inventors: Shuber, Anthony P., Lapidus, Stanley N.
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/067,212
• Time in Patent Office: 1,849 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 536/23.1, 536/24.33, 536/25.3, 536/24.3, 536/24.32, 436/94
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods are provided for selective nucleic acid sequence detection in single base primer extension reactions of high sensitivity. These methods are useful for detecting small amounts of mutant nucleic acid in a heterogeneous biological sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.

Claims

1. A method for identifying a single nucleotide in a nucleic acid sample, the method comprising the steps of:

(a) annealing an oligonucleotide primer to a nucleic acid sample under conditions that promote exact complementary hybridization between said primer and a portion of a nucleic acid in said sample;

(b) extending said primer by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;

(c) separating said extended primer from said portion;

(d) repeating steps (a) through (c) at least once; and

(e) identifying said base incorporated into said extended primer in step (b), thereby to identify said single nucleotide.

2. The method of claim 1, wherein said extension reaction in step (b) is performed in the presence of one or more chain-terminating nucleotides.

3. The method of claim 1, wherein said single base is detectably labeled.

4. The method of claim 1, wherein said single nucleotide is a polymorphic variant.

5. The method of claim 1, wherein said sample comprises nucleic acid pooled from a plurality of tissue or body fluid specimens from different individuals.

6. The method of claim 1, wherein said sample of nucleic acid is heterogeneous.

7. The method of claim 1, wherein in said annealing step an excess concentration of said oligonucleotide primer is used.

8. The method of claim 1, wherein said nucleic acid sample in step (a) has been subjected to a prior amplification step.

9. The method of claim 1, wherein, in step (a), nucleic acids in said nucleic acid sample are bound to a solid-phase support.

10. The method of claim 1, wherein, in step (a), two or more oligonucleotide primers are added, each of said two or more primers promoting hybridization to a different portion of a nucleic acid in said sample.

11. The method of claim 1, wherein, in step (b), said extension reaction is performed in the presence of four different nucleotides.

12. The method of claim 1, wherein, in step (b), said nucleotides are deoxynucleotides.

13. A method for identifying a single nucleotide in a nucleic acid sample, the method comprising the steps of:

(a) annealing an oligonucleotide primer to a nucleic acid sample, wherein said primer is a segmented primer comprising two or more probes, wherein none of said probes alone is capable of serving as a primer for template-dependent extension but wherein, when said two or more probes anneal to said nucleic acid sample adjacent to each other, they are capable of priming template-dependent extension;

(b) extending said primer by a single base;

(c) separating said extended primer from said portion;

(d) repeating steps (a) through (c) at least once; and

(e) identifying said base incorporated into said extended primer in step (b), thereby to identify said single nucleotide.

14. The method of claim 13, wherein said segmented primer comprises one probe having less than 15 nucleotides and one probe having greater than or equal to 15 nucleotides.

15. The method of claim 13, wherein said segmented primer comprises three or more probes, each said probe having fewer than 15 nucleotides.

16. A method for detecting the presence of a known single nucleotide polymorphic variant, said method comprising the steps of:

(a) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a single nucleotide polymorphic variant;

(b) extending said primer by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;

(c) separating said primer from said nucleic acid;

(d) repeating steps (a) through (c) at least once; and

(e) identifying said single base, thereby to identify the polymorphic variant.

17. The method of claim 16, wherein said single nucleotide polymorphic variant is indicative of disease.

18. The method of claim 17, wherein said disease is selected from the group consisting of cancer, cystic fibrosis, tay-sachs, sickle cell anemia, Gaucher'"'"'s disease, B-thalassemia, and parasitic infections.

19. A method for identifying a single nucleotide polymorphic variant in a population, said method comprising the steps of:

(a) obtaining a sample comprising nucleic acid from each member of a plurality of individuals in a population;

(b) annealing an oligonucleotide primer to a portion of said nucleic acid in each sample, said primer being complementary to a portion of the nucleic acid immediately upstream of a suspected single nucleotide polymorphic variant;

(c) extending said primer in each sample by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;

(d) separating said primer from said nucleic acid in each sample;

(e) repeating steps (b) through (d) at least once;

(f) identifying said single base in each sample; and

(g) identifying a single nucleotide polymorphic variant as the presence of more than one base identified in step (f).

20. The method of claim 19, wherein said samples are combined and said method is carried out on said combined samples.

21. A method for detecting the presence of a target nucleotide at a genetic locus of interest, said method comprising the steps of:

(a) obtaining a sample comprising heterogeneous nucleic acid;

(b) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a target nucleotide at a genetic locus of interest;

(c) extending said primer in the presence of a labeled nucleotide that is complementary to said target nucleotide;

(d) further extending said primer in the presence of a terminator nucleotide, thereby to generate an extension product;

(e) separating said primer from said nucleic acid;

(f) repeating steps (b) through (e) at least once; and

(g) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target nucleotide is present at said genetic locus.

22. The method of claim 21, wherein, in step (c), said labeled nucleotide is a deoxynucleotide and, in step (d), said terminator nucleotide is a dideoxynucleotide.

23. The method of claim 21, wherein, in step (c), only one species of labeled nucleotide is present.

24. A method for detecting the presence of a target single nucleotide polymorphic variant at a genetic locus of interest, said method comprising the steps of:

(a) obtaining a sample comprising heterogeneous nucleic acid;

(b) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a target single nucleotide polymorphic variant position;

(c) extending said primer in the presence of a labeled nucleotide that is complementary to a nucleotide suspected to be present at said target position;

(d) further extending said primer in the presence of an unlabeled terminator nucleotide, thereby to generate an extension product;

(e) separating said primer from said nucleic acid;

(f) repeating steps (b) through (e) at least once; and

(g) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target single nucleotide polymorphic variant is present at said genetic locus.