NUCLEIC ACID DETECTION COMBINING AMPLIFICATION WITH FRAGMENTATION

• US 20090181378A1
• Filed: 02/21/2008
• Published: 07/16/2009
• Est. Priority Date: 04/27/2007
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Bender, Richard A., Strom, Charles M., Qu, Kevin Z., Sanders, Heather R.
• Granted Patent: US 8,999,634 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Provided herein are methods and compositions for detection of a nucleic acid target in a sample. The methods and compositions use primer directed amplification in conjunction with nucleic acid fragmentation. The methods have high sensitivity even in the presence of a large amount of non-target nucleic acid. Also provided are oligonucleotides and kits useful in the method. Exemplary nucleic acid targets are those with mutant gene sequence such as mutant sequence of the EGFR, APC, TMPRSS2, ERG and ETV1 genes.

Claims

1. A method for detecting the presence or absence of a target nucleic acid by testing a sample that potentially contains the target nucleic acid in the presence of non-target nucleic acid, said method comprising:

a) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the target nucleic acid results in an increased detection of said target nucleic acid over said non-target nucleic acid as compared to amplification without fragmentation;

b) amplifying the target nucleic acid with a pair of primers, wherein a first primer is specific for the target nucleic acid; and

c) detecting the presence or absence of an amplification product, which indicates the presence or absence of the target nucleic acid in the sample.

2. The method of claim 1 wherein the first primer specific for the target nucleic acid comprises at least 5 consecutive nucleotides of sequence from non-target nucleic acid.

3. The method of claim 2 wherein the fragmentation occurs in the non-target nucleic acid at a site within the at least 5 consecutive nucleotides of sequence from said non-target nucleic acid, or complement thereof.

4. The method of claim 1 wherein the fragmentation occurs in the non-target nucleic acid at a site downstream of at least one of the primers.

5. The method of claim 1 wherein the fragmentation occurs upstream and downstream of the target nucleic acid.

6. The method of claim 1, wherein said fragmenting is achieved by digesting with a restriction enzyme.

7. The method of claim 1, wherein said target nucleic acid comprises one or more mutant sequences selected from the group consisting of insertion, deletion, substitution and translocation.

8. The method of claim 1 wherein the second of said primer pair is not specific for the target nucleic acid.

9. The method of claim 1, wherein said method further comprises nucleic acid extraction comprises one or more methods selected from the group consisting of lysis and magnetic bead extraction, phenol:

chloroform extraction, and isopropanol extraction.

10. The method of claim 1, wherein said method further comprises nucleic acid extraction using an RNA carrier.

11. The method of claim 1, wherein said target nucleic acid comprises a deletion.

12. The method of claim 11, wherein said target nucleic acid specific primer is complementary to sequence from each end of the deletion.

13. The method of claim 11, wherein said deletion is E746_A750del of the epidermal growth factor receptor (EGFR) gene.

14. The method of claim 13, wherein said fragmenting is achieved by digestion with the restriction enzyme MseI.

15. The method of claim 1, wherein said target nucleic acid comprises an insertion.

16. The method of claim 15, wherein said target specific nucleic acid primer comprises the inserted sequence or a portion thereof.

17. The method of claim 15, wherein said target specific nucleic acid primer comprises a sequence complementary to the inserted sequence or a portion thereof.

18. The method of claim 15, wherein said primer specific for a target sequence spans a junction of the inserted sequence, wherein the primer sequence includes both a portion of the original sequence and at least a portion of the inserted sequence.

19. The method of claim 15, wherein said insertion is in exon 16 of the Adenomatosis Polyposis Coli gene (APC).

20. The method of claim 15, wherein said restriction enzyme is MseI.

21. The method of claim 1, wherein said target nucleic acid comprises a substitution.

22. The method of claim 21, wherein said nucleic acid target specific nucleic acid primer comprises the substitution.

23. The method of claim 21, wherein said substitution is L858R of the EGFR gene.

24. The method of claim 21, wherein said fragmenting is achieved by digesting with the enzyme EaeI.

25. The method of claim 1, wherein said target nucleic acid comprises a chromosomal translocation.

26. The method of claim 25, wherein said target specific nucleic acid primer contains sequence that is complementary to sequence which spans a translocation junction.

27. The method of claim 25, wherein one of said primers is complementary to one chromosome of the translocation and the other primer is complementary to the other chromosome of the translocation.

28. The method of claim 25, wherein amplifying comprises one primer complementary to sequence upstream of a translocation junction and a second primer complementary to sequence downstream of the translocation junction.

29. The method of claim 25, wherein said translocation is TMPRSS2:

ERG.

30. The method of claim 25, wherein said fragmenting is achieved by digesting with the restriction enzyme FatI.

31. The method of claim 25, wherein said translocation is TMPRSS2:

ETV1.

32. The method of claim 31, wherein said fragmenting is achieved by digesting with the restriction enzyme HpyCH4V.

33. The method of claim 1, wherein said target specific nucleic acid primer is labeled with a fluorescent label.

34. The method of claim 1, wherein said sample is a tissue sample.

35. The method of claim 34, wherein said tissue sample comprises tumor tissue.

36. The method of claim 1, wherein said sample is a body fluid sample.

37. The method of claim 36, wherein said body fluid sample is a plasma sample.

38. The method of claim 1, wherein said amplifying is accomplished with polymerase chain reaction (PCR).

39. The method of claim 1, wherein said amplifying is accomplished with real time PCR(RT-PCR).

40. The method of claim 1, wherein at least one primer is a scorpion arms primer.

41. A method for diagnosing an individual with cancer by determining if an individual has a mutant sequence associated with the cancer, said method comprising:

a) obtaining a sample comprising nucleic acid from the individual;

b) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation;

c) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid; and

d) detecting the presence or absence of an amplification product containing the mutant sequence, wherein diagnosis of cancer is determined by the presence absence or amount of amplification product containing the mutant sequence.

42. The method of claim 41, wherein the cancer comprises one or more selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, colon cancer, ovarian cancer, hepatocellular cancer, and pancreatic cancer.

43. A method for prognosis of an individual diagnosed with cancer by determining if an individual has a mutant sequence associated with the cancer, said method comprising:

a) obtaining a sample comprising nucleic acid from the individual;

b) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation;

c) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid; and

d) detecting the presence, absence and/or amount of an amplification product containing the mutant sequence, wherein the likelihood of an outcome in said individual is associated with the presence and or amount of mutant nucleic acid sequence.

44. The method of claim 43, wherein the cancer comprises one or more selected from the group consisting of non-small cell lung cancer (NSCLC), breast cancer, colon cancer, ovarian cancer, hepatocellular cancer, and pancreatic cancer.

45. A method for drug sensitivity of an individual diagnosed with cancer, said method comprising:

a) obtaining a sample comprising nucleic acid from the individual;

b) fragmenting the sample nucleic acid under conditions such that a subsequent amplification directed to the mutant nucleic acid results in an increased detection of said mutant nucleic acid over said non-mutant nucleic acid as compared to amplification without fragmentation;

c) amplifying the mutant nucleic acid with a pair of primers, wherein a first primer is specific for the mutant nucleic acid;

d) detecting the presence, absence and/or amount of an amplification product containing the mutant sequence; and

e) relating the presence, absence and/or amount of an amplification product containing the mutant sequence to cancer drug sensitivity.