Multiplex amplification detection assay
Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.
1. A method of analyzing a sample for multiple target nucleic acids in a PCR-flap assay, comprising:
a) providing a sample comprising DNA suspected of containing one or more of a plurality of different target regions;
b) treating the sample in an amplification reaction mixture comprising a PCR-flap assay buffer comprising 6 to 10 mM Mg′ under conditions wherein the different target regions, if present in the sample, are amplified to form a pre-amplified mixture;
c) partitioning the pre-amplified mixture into a plurality of different PCR-flap assay reaction mixtures comprising the PCR-flap assay buffer; and
d) conducting a plurality of PCR-flap assays with the PCR-flap assay reaction mixtures, wherein the different target regions, if present in the sample at step a), are amplified and detected in the PCR-flap assay reaction mixtures.
2. The method of claim 1 , wherein the DNA is from a human subject.
3. The method of claim 1 , wherein the sample is prepared from a body fluid.
4. The method of claim 1 , wherein the body fluid comprises plasma.
5. The method of claim 1 , wherein the DNA comprises bisulfite-treated DNA.
6. The method of claim 1 , wherein the sample is prepared from cell-free DNA isolated from plasma.
7. The method of claim 1 , wherein the amplification reaction comprises at least 3 different primer pairs for amplifying at least 3 different target regions, if present in the sample.
8. The method of claim 1 , wherein the amplification reaction comprises at least 4 different primer pairs for amplifying at least 4 different target regions, if present in the sample.
9. The method of claim 1 , wherein the pre-amplified mixture is diluted with a diluent prior to partitioning.
10. The method of claim 1 , wherein the amplification reaction mixture and/or the plurality of different PCR-flap assay reaction mixtures comprise bulk fish DNA.
11. The method of claim 1 , wherein the PCR-flap assay reaction mixtures comprise a FEN-1 endonuclease.
12. The method of claim 1 , wherein the PCR-flap assay buffer comprises MgCl 2 .
13. The method of claim 1 , wherein the amplification reaction mixture comprises at least 2 different primer pairs for amplifying the at least 2 different target regions, and wherein each PCR-flap assay reaction mixture comprises an additional amount of a primer pair selected from the at least 2 different primer pairs.
14. The method of claim 13 , wherein each PCR-flap assay reaction mixture further comprises:
i) a flap oligonucleotide, and
ii) a hairpin oligonucleotide comprising a region that is complimentary to a portion of the flap oligonucleotide.
15. The method of claim 1 , wherein the amplification reaction mixture of step b) is exposed to thermal cycling conditions limited to fewer than 20 thermal cycles.
16. The method of claim 15 , wherein the hairpin oligonucleotide comprises a fluorophore moiety.
17. The method of claim 1 , wherein the plurality of different target regions comprises a reference target region.
18. The method of claim 17 , wherein said reference target region comprises β-actin or ZDHHC1.
19. The method of claim 1 , wherein the sample comprises a DNA process control.
20. The method of claim 19 , wherein the DNA process control comprises a zebrafish RASSF1 sequence.