Isolation and detection of dna from plasma

Patent metadata

Field	Value
Publication number	US-2023392189-A1
Application number	US-202318310276-A
Country	US
Kind code	A1
Filing date	May 1, 2023
Priority date	Oct 30, 2015
Publication date	Dec 7, 2023
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 .- 21 . (canceled)

22 . A method of processing a sample, the method comprising:
a) combining a sample comprising nucleic acid with:
i) a first portion of guanidine thiocyanate; and
ii) a first portion of non-ionic detergent; and
iii) exogenous non-target DNA;
to form a first mixture; b) to the first mixture, 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 nucleic acid from said sample is bound to the silica particles.

23 . The method of claim 22 , further comprising:
c) separating silica particles with bound nucleic acid from the second mixture.

24 . The method of claim 23 , further comprising:
d) washing separated silica particles with bound nucleic acid with a wash solution comprising alcohol.

25 . The method of claim 24 , comprising washing separated silica particles with bound nucleic acids with a first wash solution comprising guanidine hydrochloride or guanidine thiocyanate, and ethyl alcohol.

26 . The method of claim 25 , further comprising washing separated silica particles with bound nucleic acid with a second wash solution comprising a buffer and ethyl alcohol.

27 . The method of claim 24 , further comprising eluting nucleic acid from washed separated silica particles with bound nucleic acid to produce eluted nucleic acid.

28 . The method of claim 22 , 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).

29 . The method of claim 22 , wherein the sample is blood or a blood product.

30 . The method of claim 29 , wherein the sample is a blood product comprising plasma and/or serum.

31 . The method of claim 22 , wherein the sample is from a human subject.

32 . The method of claim 31 , wherein the exogenous non-target DNA comprises genomic DNA from a non-human source.

33 . The method of claim 32 , wherein the genomic DNA is from fish.

34 . The method of claim 22 , wherein the first mixture comprises a protease.

35 . The method of claim 34 , wherein the protease comprises Proteinase K.

36 . The method of claim 27 , further comprising treating at least a portion of the eluted nucleic acid with a bisulfite reagent.

37 . The method of claim 27 , further comprising treating at least a portion of the eluted nucleic acid in an amplification reaction mixture that comprises the portion of eluted nucleic acid and amplification reagents to produce amplified DNA, wherein the portion of eluted nucleic acid used in the amplification reaction mixture has a volume and the amplification reaction mixture has a total volume, and wherein the volume of the portion of eluted nucleic acid used in the amplification reaction mixture is at least 20% of the total volume of the amplification reaction mixture.

38 . The method of claim 37 , wherein the volume of the eluted nucleic acid used in the amplification reaction mixture is at least 50% of the total volume of the amplification reaction mixture.

39 . The method of claim 37 , wherein the amplification reaction mixture comprises a buffer comprising 3-(n-morpholino) propanesulfonic acid (MOPS) buffer and 6 to 10 mM Mg ++ .

Assignees

• Exact Sciences Corp

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