Multiplex amplification detection assay

Abstract

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 - 22 . (canceled) 
     
     
         23 . A method of analyzing a sample in a PCR-flap assay, comprising:
 a) providing a sample comprising DNA suspected of containing at least one target region of a plurality of different target regions;   b) treating the sample in an amplification reaction mixture comprising a PCR-flap assay buffer comprising 3-(n-morpholino) propanesulfonic acid (MOPS) buffer and 6 to 10 mM Mg ++  under conditions wherein the at least one target region, if present in the sample, is amplified to form a pre-amplified mixture;   c) partitioning at least one portion of the pre-amplified mixture into at least one PCR-flap assay reaction mixture comprising the PCR-flap assay buffer; and   d) conducting at least one PCR-flap assay with the at least one PCR-flap assay reaction mixture, wherein the at least one target region, if present in the sample at step a), is amplified and detected in the at least one PCR-flap assay reaction mixture.   
     
     
         24 . The method of  claim 23 , wherein the at least one target region comprises a plurality of different target regions, wherein the pre-amplified mixture is partitioned into a plurality of different PCR-flap assay reaction mixtures, and wherein the plurality of different target regions, if present in the sample at step a), are amplified and detected in the plurality of different PCR-flap assay reaction mixtures. 
     
     
         25 . The method of  claim 23 , wherein the PCR-flap assay buffer comprises MgCl 2 . 
     
     
         26 . The method of  claim 23 , wherein the PCR-flap assay buffer comprises 7.5 mM Mg ++ . 
     
     
         27 . The method of  claim 23 , wherein the PCR-flap assay buffer comprises 10 mM MOPS and 7.5 mM MgCl 2 . 
     
     
         28 . The method of  claim 23 , wherein the PCR-flap assay buffer further comprises one or more components selected from KCl, bovine serum albumin (BSA), Polyoxyethylenesorbitan monolaurate (Tween-20), and Octylphenoxy poly(ethyleneoxy)ethanol, branched (IGEPAL CA630). 
     
     
         29 . The method of  claim 28 , wherein the PCR-flap assay buffer comprises 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 7.5 mM MgCl 2 , 0.8 mM KCl, 0.1 μg/μL BSA, 0.0001% Tween-20 and 0.0001% IGEPAL CA-630. 
     
     
         30 . The method of  claim 23 , wherein the sample is from a human subject. 
     
     
         31 . The method of  claim 23 , wherein the sample is prepared from a body fluid. 
     
     
         32 . The method of  claim 31 , wherein the body fluid comprises plasma. 
     
     
         33 . The method of  claim 23 , wherein the sample comprises cell-free DNA isolated from plasma. 
     
     
         34 . The method of  claim 23 , wherein the DNA comprises bisulfate-treated DNA. 
     
     
         35 . The method of  claim 23 , wherein the amplification reaction mixture comprises at least 3 different primer pairs for amplifying at least 3 different target regions of the plurality of different target regions, if present in the sample. 
     
     
         36 . The method of  claim 23 , wherein the pre-amplified mixture is diluted with a diluent prior to partitioning. 
     
     
         37 . The method of  claim 23 , wherein the amplification reaction mixture and/or the plurality of different PCR-flap assay reaction mixtures comprise bulk fish DNA. 
     
     
         38 . The method of  claim 23 , wherein the PCR-flap assay reaction mixture comprises a FEN-1 endonuclease. 
     
     
         39 . The method of  claim 38 , wherein the 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.   
     
     
         40 . The method of  claim 39 , wherein the hairpin oligonucleotide comprises a fluorophore moiety. 
     
     
         41 . The method of  claim 23 , wherein the amplification reaction mixture of step b) is exposed to thermal cycling conditions limited to fewer than 20 thermal cycles. 
     
     
         42 . The method of  claim 23 , wherein the amplification reaction mixture of step b) is exposed to thermal cycling conditions limited to 10 or fewer thermal cycles.