Methods and compositions for enrichment of nucleic acids in mixtures of highly homologous sequences

• US 9,487,824 B2
• Filed: 06/28/2012
• Issued: 11/08/2016
• Est. Priority Date: 06/28/2011
• Status: Active Grant

• Current Assignee: Kutyavin, Igor
• Original Assignee: Kutyavin, Igor
• Inventors: Kutyavin, Igor
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US14/129,268
• Publication Number:
  US 20140206001A1
• Time in Patent Office: 1,594 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2561/109 Invader technology

Abstract

Provided are methods of enrichment and detection of target nucleic acids during target amplification in the presence of excess amounts of highly homologous sequences, the methods having substantial diagnostic utility (e.g., cancer diagnostics). Provided are amplification reaction mixtures having at least one cleavage-directing oligonucleotide, the respective binding sites of which, for the target and homologous sequences, include one or more nucleotide positions differing in sequence between the target homologous sequences. During target sequence amplification in the presence of DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5′-triphosphates and other reagents suitable to support amplification of both target and homologous nucleic acid sequences, the cleavage directing oligonucleotide hybridizes to the homologous sequence and its amplification products resulting in a FEN1-mediated cleavage of these sequences providing for increased amounts of the target nucleic acid sequence relative to that of the homologous nucleic acid sequences.

Claims

1. A method for enrichment during amplification of a target nucleic acid sequence in a mixture of target and homologous nucleic acid sequences, the method comprising:

a) providing an amplification reaction mixture having a target nucleic acid sequence and at least one other nucleic acid sequence that is homologous to the target nucleic acid sequence but differs therefrom in at least one nucleotide position, at least one cleavage-directing oligonucleotide sequence, the respective binding sites of which to said target and to said homologous nucleic acid sequences include the at least one nucleotide position having the nucleotide sequence difference, a DNA polymerase activity, a FEN1 activity, one or more amplification primers, deoxynucleoside 5′

-triphosphates and other reagents suitable to support the amplification of both target and homologous nucleic acid sequences; and

b) amplifying the nucleic acids under reaction conditions suitable to support hybridization of the cleavage-directing oligonucleotide sequence to the homologous nucleic acid sequence providing for FEN1-mediated cleavage of the homologous nucleic acid and its amplification products, to provide for preferential amplification and increase in amount of the target nucleic acid sequence relative to that of the homologous nucleic acid sequence.

2. The method of claim 1, comprising, prior to amplifying, rendering the target nucleic acid and the at least one homologous nucleic acid single-stranded;

and incubating the single-stranded target and homologous nucleic acids in the presence of the FEN1 activity and the at least one cleavage-directing oligonucleotide sequence under reaction conditions suitable to support hybridization of the cleavage-directing oligonucleotide sequence to the homologous nucleic acid sequence to provide for cleavage of the homologous nucleic acid by the FEN1 activity.

3. The method of claim 1, wherein two cleavage-directing oligonucleotide sequences are used, which hybridize to opposite strands of said homologous nucleic acid.

4. The method of claim 1, wherein the one or more amplification primers include a specialty 5′

flap sequence that is complementary to the at least one cleavage-directing oligonucleotide sequence, and wherein the cleavage-directing oligonucleotide sequence is synthesized at the 3′

-end of one or more amplification products of the target and homologous nucleic acids.

5. The method of claim 1, comprising use of a plurality of cleavage-directing nucleic acid sequences to provide for enrichment of a plurality of target nucleic acid sequences relative to a respective plurality of homologous nucleic acid sequences in the same reaction mixture.

6. The method of claim 1, wherein the cleavage-directing oligonucleotide sequence comprises, or is modified during amplification to include a duplex-stabilizing and/or a 5′

-nuclease-protecting modification.

7. The method of claim 4, comprising incorporating at least one duplex-stabilizing nucleoside modification into the at least one cleavage-directing oligonucleotide sequence during amplification using a corresponding duplex-stabilizing base-modified deoxynucleoside 5′

-triphosphate.

8. The method of claim 7, wherein the specialty 5′

flap sequence comprises, or is modified during amplification to incorporate at least one duplex-destabilizing polymerase-efficient nucleoside modification.

9. The method of claim 1 providing for an enrichment of the target nucleic acid sequence, relative to the at least one homologous nucleic acid sequence, in the range of 10³ to 10⁷-fold.

10. The method of claim 1, wherein the FEN1 activity and the DNA polymerase activity are provided by the same enzyme.

11. The method of claim 1, wherein the target nucleic acid is detected using a post-amplification method and/or a real time method.

12. The method of claim 11, further comprising determining the amount of the target nucleic acid in the sample.

13. The method of claim 1, wherein at least one of the amplification primers, amplification products and cleavage-directing oligonucleotide sequence is immobilized on a solid support during the amplification and target enrichment.

14. The method of claim 1, wherein the amplification reaction comprises PCR.

15. The method of claim 1, wherein the amplification reaction comprises an isothermal amplification reaction.

16. The method of claim 11, wherein detecting the target nucleic acid comprises use of an oligonucleotide probe.

17. The method of claim 12, wherein detecting the target nucleic acid comprises use of an oligonucleotide probe.

18. The method of claim 17, wherein the oligonucleotide probe is fluorescently labeled.

19. The method of claim 18, wherein the oligonucleotide probe comprises a probe selected from the group consisting of a FRET probe, a cleavable FRET probe, and a FEN1-cleavable FRET probe.

20. The method of claim 19, wherein the probe detection method comprises a 5′

-nuclease assay and/or a method comprising folding of PCR amplicons into secondary structures.

21. The method of claim 1, wherein the FEN1 activity and the DNA polymerase activity are provided by different enzymes.