Nucleic acid detection combining amplification with fragmentation

• US 9,783,854 B2
• Filed: 04/06/2015
• Issued: 10/10/2017
• 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): PRIEST, AARON A
• Application Number:
  US14/679,403
• Publication Number:
  US 20150299811A1
• Time in Patent Office: 918 Days
• Field of Search: None
• 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 aligned nucleotide positions and the non-target nucleic acid comprises at least one 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 specific for the target nucleic acid, wherein one primer of the pair spans or overlaps the at least one fragmentation site present in the non-target nucleotide sequence but absent from the target nucleotide sequence, and wherein fragmentation of the non-target nucleotide sequence in part a) prevents hybridization of said 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 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 the each primer of the primer pair comprises at least 5 consecutive nucleotides of sequence from non-target nucleic acid.

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

4. 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.

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

chloroform extraction, and isopropanol extraction.

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

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

8. The method of claim 7, wherein said primer that spans or overlaps the at least one fragmentation site is complementary to sequence from each end of the deletion.

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

10. The method of claim 9, wherein said fragmenting is achieved by digestion with restriction enzyme Msel.

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

12. The method of claim 11, wherein said target specific nucleic acid primer comprises the insertion or a portion thereof.

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

14. The method of claim 11, wherein said primer specific for a target sequence spans a junction of the insertion, wherein the primer sequence includes both a portion of sequence adjacent to the insertion and at least a portion of the insertion.

15. The method of claim 11, wherein said insertion is in exon 16 of the Adenomatosis Polyposis Coli gene (APC).

16. The method of claim 11, wherein said fragmenting is achieved by digestion with restriction enzyme Msel.

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

18. The method of claim 17, wherein said nucleic acid target specific nucleic acid primer comprises the substitution.

19. The method of claim 17, wherein said substitution results in an L858R amino acid substitution of the EGFR gene.

20. The method of claim 17, wherein said fragmenting is achieved by digesting with restriction enzyme Eael.

21. The method of claim 1, wherein said target nucleic acid comprises a chromosomal translocation between a first chromosome and a second chromosome.

22. The method of claim 21, wherein said target specific nucleic acid primer contains sequence that is complementary to sequence which spans a translocation junction.

23. The method of claim 21, wherein one of said primers of the pair is complementary to the first chromosome of the translocation and one of said primers of the pair is complementary to the second chromosome of the translocation.

24. The method of claim 21, 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.

25. The method of claim 21, wherein said translocation is TMPRSS2:

ERG.

26. The method of claim 21, wherein said fragmenting is achieved by digesting with restriction enzyme Fatl.

27. The method of claim 21, wherein said translocation is TMPRSS2:

ETV1.

28. The method of claim 27, wherein said fragmenting is achieved by digesting with restriction enzyme HpyCH4V.

29. The method of claim 1, wherein one or both primers of the primer pair is labeled with a fluorescent label.

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

31. The method of claim 30, wherein said tissue sample comprises tumor tissue.

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

33. The method of claim 32, wherein said body fluid sample is a plasma sample.

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

35. 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 specific for the target nucleotide sequence, wherein one primer spans or overlaps the at least one fragmentation site present in the non-target nucleotide sequence but absent from the target nucleotide sequence, and wherein fragmentation of the non-target nucleotide sequence in part a) prevents hybridization of said 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 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.