Isolation and detection of dna from plasma

Patent metadata

Field	Value
Publication number	US-2021079451-A1
Application number	US-202017031383-A
Country	US
Kind code	A1
Filing date	Sep 24, 2020
Priority date	Oct 30, 2015
Publication date	Mar 18, 2021
Grant date	—

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.

First claim

1 . A method of processing a plasma sample, the method comprising:
a) combining a plasma sample with
i) protease;
ii) a first portion of guanidine thiocyanate; and
iii) a first portion of non-ionic detergent,
to form a first mixture wherein proteins are digested by said protease; b) to the first mixture of step a) adding
iv) silica particles;
v) isopropyl alcohol;
vi) a second portion of guanidine thiocyanate; and
vii a second portion of non-ionic detergent,
to form a second mixture under conditions wherein DNA is bound to said silica particles; c) separating silica particles with bound DNA from the second mixture; 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 the washed silica particles with bound DNA from said second wash solution; h) eluting DNA from said washed silica particles with bound DNA 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 first portion and the second portion of non-ionic detergent 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 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 first mixture 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 portion of guanidine thiocyanate and said first portion of non-ionic detergent are added to the plasma sample together as a first lysis reagent comprising about 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630 and/or wherein said second portion of guanidine thiocyanate and said second portion of non-ionic detergent are added to the first mixture together as a second lysis reagent comprising 4.3 M guanidine thiocyanate and 10% w:v IGEPAL CA-630 combined with isopropyl alcohol.

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.

14 . A kit for isolating DNA from plasma, the kit comprising:
a) a first lysis reagent comprising guanidine thiocyanate and non-ionic detergent, or components for preparing said first lysis reagent; b) a second lysis reagent comprising guanidine thiocyanate, non-ionic detergent, and isopropanol, or components for preparing said second lysis reagent; c) a first wash solution comprising guanidine hydrochloride or guanidine thiocyanate and ethyl alcohol, or components for preparing said first wash solution; d) a second wash solution comprising Tris buffer and ethyl alcohol, or components for preparing said second wash solution; and e) silica particles.

15 . The kit of claim 14 , 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).

16 . The kit of claim 14 , further comprising one or more of:
i) an elution buffer or components for preparing said elution buffer; ii) a DNA process control; and/or iii) a preparation of bulk fish DNA.

17 . The kit of claim 16 , wherein said DNA process control comprises a zebrafish RASSF1 sequence.

18 . A system for processing a plasma sample, the system comprising:
a) a first lysis reagent comprising guanidine thiocyanate and non-ionic detergent, or components for preparing said first lysis reagent; b) a second lysis reagent comprising guanidine thiocyanate, non-ionic detergent, and isopropanol, or components for preparing said second lysis reagent; c) a first wash solution comprising guanidine hydrochloride or guanidine thiocyanate and ethyl alcohol, or components for preparing said first wash solution; d) a second wash solution comprising Tris buffer and ethyl alcohol, or components for preparing said second wash solution; and e) silica particles.

19 . The system of claim 18 , 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).

20 . The system of claim 18 , further comprising one or more of:
i) a protease; ii) an elution buffer or components for preparing said elution buffer; iii) a DNA process control; iv) a preparation of bulk fish DNA; v) reagents for treating DNA to produce bisulfite-treated DNA.

21 . The system of claim 18 , further comprising PCR amplification reagents and/or PCR flap assay reagents, wherein said PCR amplification reagents comprise:
i) a plurality of different primer pairs for amplifying a plurality of different target regions, if present in said plasma; ii) thermostable DNA polymerase; iii) dNTPs; and iv) a buffer comprising Mg ++ ;
and wherein said PCR flap assay reagents comprise:
i) a plurality of different primer pairs for amplifying a plurality of different target regions, if present in said plasma;
ii) thermostable DNA polymerase;
iii) dNTPs;
iv) a buffer comprising Mg ++
v) a flap endonuclease;
vi) a flap oligonucleotide, and
vii) a hairpin oligonucleotide comprising a region that is complimentary to a portion of said flap oligonucleotide.

Assignees

• Exact Sciences Dev Co Llc

Inventors

• Allawi Hatim
• Lidgard Graham P
• Aizenstein Brian
• Sander Tamara J
• Giakoumopoulos Maria
• Kaiser Michael W
• Gray Melissa M
• Vaccaro Abram Michael

Classifications

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title