Multiplex amplification detection assay II

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 the 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 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 comprise a target-specific region of at least 13 bases in length and further comprise one or more nucleotides that are capable of making non-Watson-Crick base pairs.

3. The method of claim 2 , wherein the one or more nucleotides that are capable of making non-Watson-Crick base pairs comprise inosine.

4. The method of claim 1 , wherein the average size of the amplicons in the pre-amplified mixture is 50 to 85 base pairs.

5. The method of claim 1 , wherein step (a) comprises 10 amplification cycles.

6. The method of claim 1 , wherein the PCR-flap assays further comprise an invasive oligonucleotide that together with a target region and a flap oligonucleotide forms a substrate cleaved by a flap endonuclease.

7. The method of claim 1 , wherein the detection assays comprise a denaturation step at a temperature that is at least 90° C., an annealing step at a temperature that is in the range of 60° C. to 65° C., and an extension step at a temperature in the range of 65° C. to 75° C.

8. The method of claim 1 , wherein said bisulfite treated DNA is from a human subject.

9. The method of claim 8 , wherein said sample is prepared from a body fluid.

10. The method of claim 9 , wherein said body fluid comprises plasma.

11. The method of claim 10 , wherein the sample is prepared from cell-free DNA isolated from plasma.

12. The method of claim 11 , wherein said cell-free DNA is less than 200 base pairs in length.

13. The method of claim 11 , 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.

14. The method of claim 13 , wherein said protease is Proteinase K.

15. The method of claim 9 , wherein the sample is prepared from at least one mL of bodily fluid.

16. The method of claim 1 , wherein the pre-amplified mixture is partitioned into at least 4 detection assay reaction mixtures.