Multiplex amplification detection assay II
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, comprising:
(a) amplifying a sample comprising bisulfite-treated DNA by PCR in a single reaction using a plurality of different primer pairs to produce a pre-amplified mixture;
(b) partitioning the pre-amplified mixture into a plurality of different detection assay reaction mixtures, wherein each detection assay reaction mixture comprises a portion of said pre-amplified mixture; and
(c) conducting a plurality of detection assays on the detection assay reaction mixtures, wherein the detection assays are PCR-flap assays that employ flap oligonucleotides that have a target-specific region of at least 13 bases in length and an additional amount of a primer pair selected from said plurality of different primer pairs of step (a), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair.
2. The method of claim 1 , wherein one or more of the flap oligonucleotides used in (c) has a target-specific region comprising one or more nucleotides that are capable of making non-Watson-Crick base pairs.
3. The method of claim 1 , wherein one or more of the flap oligonucleotides used in (c) has a target-specific region having a length in the range of 13 to 30 bases.
4. The method of claim 1 , wherein the one or more of the flap oligonucleotides used in (c) has a target-specific region that has a T m in the range of 60° C. to 70° C. and the detection assays comprise a denaturation step at least 90° C., an annealing step at a temperature that is in the range of 60° C. to 70° C., and an extension step at a temperature in the range of 65° C. to 75° C.
5. The method of claim 1 , wherein said bisulfite treated DNA is from a human subject.
6. The method of claim 5 , wherein said sample is prepared from a body fluid.
7. The method of claim 6 , wherein said body fluid comprises plasma.
8. The method of claim 7 , wherein the sample is prepared from cell-free DNA isolated from plasma.
9. The method of claim 8 , wherein said cell-free DNA is less than 200 base pairs in length.
10. The method of claim 8 , wherein said cell-free DNA is isolated from said plasma by a method comprising:
a) combining the plasma sample with:
i) protease; and
ii) a first lysis reagent, said first lysis reagent comprising
guanidine thiocyanate; and
non-ionic detergent;
to form a mixture wherein proteins are digested by said protease;
b) to the mixture of step a) adding
iii) silica particles, and
iv) a second lysis reagent, said second lysis reagent comprising:
guanidine thiocyanate;
non-ionic detergent; and
isopropyl alcohol;
under conditions wherein DNA is bound to said silica particles;
c) separating silica particles with bound DNA from the mixture of b);
d) to the separated silica particles with bound DNA adding a first wash solution, said first wash solution comprising guanidine hydrochloride or guanidine thiocyanate and ethyl alcohol;
e) separating the silica particles with bound DNA from said first wash solution;
f) to the separated silica particles with bound DNA adding a second wash solution, said second wash solution comprising a buffer and ethyl alcohol;
g) separating washed silica particles with bound DNA from said second wash solution; and
h) eluting DNA from the washed silica particles with bound DNA.
11. The method of claim 10 , wherein said protease is Proteinase K.
12. The method of claim 6 , wherein the sample is prepared from at least one mL of bodily fluid.
13. The method of claim 1 , wherein the pre-amplified mixture is partitioned into at least 4 detection assay reaction mixtures.