Nucleic acid detection combining amplification with fragmentation

• US 8,999,634 B2
• Filed: 02/21/2008
• Issued: 04/07/2015
• 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: Sanders, Heather R., Qu, Kevin Z., Strom, Charles M., Bender, Richard A.
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US12/035,356
• Publication Number:
  US 20090181378A1
• Time in Patent Office: 2,602 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 containing unamplified nucleic acids that potentially contains the target nucleic acid in the presence of non-target nucleic acid, said method comprising:

a) fragmenting the unamplified nucleic acids under conditions such that the non-target nucleic acid is preferentially fragmented relative to the target nucleic acid, wherein the target nucleic acid and the non-target nucleic acid are at least 50% identical at the aligned nucleotide positions and the non-target nucleic acid comprises a fragmentation site not present in the target nucleic acid;

b) combining the unamplified nucleic acids of step a) with a polymerase chain reaction (PCR) mixture comprising a pair of primers selected from the group consisting of SEQ ID NOs;

1 and 2, SEQ ID NOs;

3 and 4, SEQ ID NOs;

5 and 6, SEQ ID NOs;

7 and 8, SEQ ID NOs;

9 and 10, and SEQ ID NOs;

11 and 12, wherein fragmentation of the non-target nucleotide sequence in part a) prevents hybridization of one primer to the non-target nucleic acid;

c) amplifying the target nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step b); and

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

wherein the non-target nucleic acid comprises a wild-type nucleotide sequence and the target nucleic acid comprises a mutant version of the wild-type nucleotide sequence, and wherein step a) is performed prior to step b).

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

3. The method of claim 1, wherein said mutant version of the wild-type nucleotide sequence comprises one or more sequence mutations selected from the group consisting of an insertion, deletion, substitution and translocation as compared to the wild-type nucleotide sequence.

4. 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.

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

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

7. The method of claim 6, wherein said deletion is E746_A750del of the epidermal growth factor receptor (EGFR) gene.

8. The method of claim 7, wherein said fragmenting is achieved by digestion with the restriction enzyme MseI.

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

10. The method of claim 9, wherein said target specific nucleic acid primer comprises the inserted sequence or a portion thereof.

11. The method of claim 9, wherein said target specific nucleic acid primer comprises a sequence complementary to the inserted sequence or a portion thereof.

12. The method of claim 9, 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.

13. The method of claim 9, wherein said insertion is in exon 16 of the Adenomatosis Polyposis Coli gene (APC).

14. The method of claim 9, wherein said restriction enzyme is MseI.

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

16. The method of claim 15, wherein said nucleic acid target specific nucleic acid primer comprises the substitution.

17. The method of claim 15, wherein said substitution is L858R of the EGFR gene.

18. The method of claim 15, wherein said fragmenting is achieved by digesting with the enzyme EaeI.

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

20. The method of claim 19, wherein said target specific nucleic acid primer contains sequence that is complementary to sequence which spans a translocation junction.

21. The method of claim 19, 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.

22. The method of claim 19, wherein said amplifying comprises one primer complementary to sequence upstream of a translocation junction and a second primer complementary to sequence downstream of the translocation junction.

23. The method of claim 19, wherein said translocation is TMPRSS2:

ERG.

24. The method of claim 19, wherein said fragmenting is achieved by digesting with the restriction enzyme FatI.

25. The method of claim 19, wherein said translocation is TMPRSS2:

ETV1.

26. The method of claim 25, wherein said fragmenting is achieved by digesting with the restriction enzyme HpyCH4V.

27. The method of claim 1, wherein one or both of said primers labeled with a fluorescent label.

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

29. The method of claim 28, wherein said tissue sample comprises tumor tissue.

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

31. The method of claim 30, wherein said body fluid sample is a plasma sample.

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

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

a) obtaining a sample comprising unamplified nucleic acids from the individual, wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks a fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at the aligned nucleotide positions;

b) fragmenting the unamplified nucleic acids 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) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture;

d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers selected from the group consisting of SEQ ID NOs;

1 and 2, SEQ ID NOs;

3 and 4, SEQ ID NOs;

5 and 6, SEQ ID NOs;

7 and 8, SEQ ID NOs;

9 and 10, and SEQ ID NOs;

11 and 12, wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of one primer to the non-mutant nucleic acid; and

e) detecting the presence or absence of an amplification product from step d) containing the mutant nucleic acid, wherein diagnosis of cancer is determined by the presence absence or amount of amplification product containing the mutant sequence;

wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutated version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c).

34. The method of claim 33, 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.

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

a) obtaining a sample comprising unamplified nucleic acids from the individual wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks a fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at the aligned nucleotide positions;

b) fragmenting the unamplified nucleic acids 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) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture;

d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers selected from the group consisting of SEQ ID NOs;

1 and 2, SEQ ID NOs;

3 and 4, SEQ ID NOs;

5 and 6, SEQ ID NOs;

7 and 8, SEQ ID NOs;

9 and 10, and SEQ ID NOs;

11 and 12, wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of one primer to the non-mutant nucleic acid; and

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

wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutant version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c).

36. The method of claim 35, 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.

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

a) obtaining a sample comprising unamplified nucleic acids from the individual wherein said unamplified nucleic acids potentially contain the mutant nucleic acid in the presence of non-mutant nucleic acid, wherein said mutant nucleic acid lacks a fragmentation site present in a non-mutant nucleic acid and said mutant nucleic acid and said non-mutant nucleic acid are at least 50% identical at the aligned nucleotide positions;

b) fragmenting the unamplified nucleic acids 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) combining the unamplified nucleic acids of step b) with a polymerase chain reaction (PCR) mixture;

d) amplifying the mutant nucleic acid from the unamplified nucleic acids combined with the PCR mixture in step c) with a pair of primers selected from the group consisting of SEQ ID NOs;

1 and 2, SEQ ID NOs;

3 and 4, SEQ ID NOs;

5 and 6, SEQ ID NOs;

7 and 8, SEQ ID NOs;

9 and 10, and SEQ ID NOs;

11 and 12, wherein fragmentation of the non-mutant nucleotide sequence in part b) prevents hybridization of one primer to the non-mutant nucleic acid; and

e) detecting the presence, absence and/or amount of an amplification product from step d) containing the mutant nucleic acid; and

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

wherein the non-mutant nucleic acid comprises a wild-type nucleotide sequence and the mutant nucleic acid comprises a mutant version of the wild-type nucleic acid sequence, and wherein step b) is performed prior to step c.

38. A method for detecting the presence or absence of a target nucleotide sequence in a sample potentially comprising both target and non-target nucleotide sequences, wherein the non-target nucleotide sequence comprises a wild-type nucleotide sequence, and the target nucleic acid comprises a mutant version of the wild-type nucleotide sequence lacking at least one fragmentation site present in the wild-type nucleotide sequence, and wherein the sample comprises only unamplified nucleic acids, comprising:

a) fragmenting the nucleic acids in the sample under conditions such that fragmentation occurs at the at least one fragmentation site present in the non-target nucleotide sequence but absent from the target nucleotide sequence;

b) subsequent to step a), combining the product of a) with a polymerase chain reaction (PCR) mixture comprising a pair of primers selected from the group consisting of SEQ ID NOs;

1 and 2, SEQ ID NOs;

3 and 4, SEQ ID NOs;

5 and 6, SEQ ID NOs;

7 and 8, SEQ ID NOs;

9 and 10, and SEQ ID NOs;

11 and 12, wherein fragmentation of the non-target nucleotide sequence in part a) prevents hybridization of one primer to the non-target nucleotide sequence;

c) subsequent to step b), performing an amplification reaction on the PCR mixture of b); and

d) detecting the presence or absence of an amplification product in c), wherein the presence of an amplification product in d) indicates the presence of the target nucleotide sequence in the sample.