Isolation and detection of DNA from plasma

1. A method of processing a plasma sample, the method comprising:

a) combining a plasma sample with

i) protease;

ii) a first lysis reagent, said first lysis reagent comprising

guanidine thiocyanate,

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,

iv) a second lysis reagent, said second lysis reagent comprising:

guanidine thiocyanate;

non-ionic detergent;

and

v) 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 from step c) 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 from step e) adding a second wash solution, said second wash solution comprising a buffer and ethyl alcohol;

g) separating the washed silica particles with bound DNA from said second wash solution;

h) eluting DNA from said washed silica particles with bound DNA from step g) to produce an isolated DNA sample.

2. The method of claim 1 , wherein steps f)-g) are repeated 1 to 3 additional times prior to step h).

3. The method of claim 1 , wherein the non-ionic detergents in the first lysis reagent and the second lysis reagent are the same or different, and are selected from the group consisting of polyethylene glycol sorbitan monolaurate (Tween-20), octylphenoxypolyethoxyethanol (Nonidet P-40), and octylphenoxy poly(ethyleneoxy) ethanol, branched (IGEPAL CA-630).

4. The method of claim 1 , wherein said protease is Proteinase K protease.

5. The method of claim 1 , further comprising a step after step g) and prior to step h) of drying the washed silica particles with bound DNA.

6. The method of claim 1 , wherein the mixture of step a) further comprises a DNA process control.

7. The method of claim 6 , wherein said DNA process control comprises a zebrafish Ras association domain (RalGDS/AF-6) family 1 (RASSF1) sequence.

8. The method of claim 7 , wherein said DNA process control comprising a zebrafish RASSF1 sequence is synthetic DNA.

9. The method of claim 1 , wherein the mixture of step a) further comprises bulk fish DNA.

10. The method of claim 1 , wherein said plasma sample has a volume of at least 2 mL.

11. The method of claim 1 , wherein said first lysis reagent comprises about 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630 and/or wherein said second lysis reagent comprises 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630 and is combined with the isopropyl alcohol prior to step b).

12. The method of claim 1 , wherein said first wash solution comprises about 3 M guanidine hydrochloride or 3 M guanidine thiocyanate and about 57% ethyl alcohol and/or said second wash solution comprises about 80% ethyl alcohol and about 20% 10 mM Tris pH 8.0 buffer.

13. The method of claim 1 , further comprising analyzing said isolated DNA sample for multiple target nucleic acids, comprising:

a) treating said isolated DNA sample with bisulfite to produce a bisulfite treated DNA sample;

b) treating said bisulfite-treated DNA sample to an amplification reaction under conditions wherein at least five different target regions, if present in said sample, are amplified to form a pre-amplified mixture;

c) partitioning said pre-amplified mixture into a plurality of different detection assay reaction mixtures; and

d) conducting a plurality of detection assays with said detection assay reaction mixtures, wherein said at least five different target regions, if present in said sample at step a), are detected in one or more of said plurality of different detection assay reaction mixtures.