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US 10,648,025 B2Granted
Multiplex amplification detection assay II
AssigneeExact Sciences Development Company, LLC
InventorsHatim T. Allawi, William G. Weisburg, Graham P. Lidgard, Michael W. Kaiser, Abram M. Vaccaro, Gracie Shea
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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.
Claims20
A method of analyzing a sample for multiple target nucleic acids, comprising:
- a) providing a sample having volume x, the sample comprising bisulfite-treated DNA suspected of containing one or more of a plurality of n different target regions that are in different fragments in the sample, wherein n is at least 3, and wherein at least one of said target regions is a low-copy target that, if present in said sample, is present in said sample at a copy number such that:
- i) among n fractions of said sample each having a volume of x/n, said low copy target is absent from one or more of said n fractions, or
- ii) among n fractions of said sample each having a volume of x/n, said low copy target in one or more of said n fractions is below a level of sensitivity of a detection assay for said low copy target, wherein the detection assay is a PCR-flap assay without a pre-amplification step;
- b) treating said volume x of said sample to an amplification reaction using a plurality of different primer pairs for amplifying n different target regions, if present in said sample under conditions wherein said n different target regions, if present in said sample, are amplified to form a pre-amplified mixture having volume y;
- c) partitioning said 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 that has a volume of y/n or less;
- d) conducting a plurality of detection assays with said detection assay reaction mixtures, wherein said different target regions, if present in said sample at step a), are detected in said 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 b), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair.
3 more elements
A method for analyzing multiple target nucleic acids in a sample using a PCR pre-amplification and a PCR-flap assay, the method comprising:
- a) providing bisulfite-treated DNA comprising a plurality of different target regions in a first reaction mixture comprising PCR amplification reagents, wherein said PCR amplification reagents comprise:
- i) a plurality of different primer pairs for amplifying said plurality of different target regions, if present in said sample, from said bisulfite-treated DNA;
- ii) thermostable DNA polymerase;
- iii) dNTPs;
- and
- iv) a buffer comprising Mg++
- b) exposing said first reaction mixture to thermal cycling conditions wherein a plurality of different target regions, if present in the sample, are amplified to produce a pre-amplified mixture, and wherein said thermal cycling conditions are limited to fewer than 20 thermal cycles;
- c) partitioning said pre-amplified mixture into a plurality of PCR-flap assay reaction mixtures, wherein each PCR-flap assay reaction mixture comprises:
- i) an additional amount of a primer pair selected from said plurality of different primer pairs of step a) i), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair;
- ii) thermostable DNA polymerase;
- iii) dNTPs;
- iv) said buffer comprising Mg++
- v) a flap endonuclease;
- vi) a flap oligonucleotide that has a target-specific region of at least 13 bases in length, and
- vi) a hairpin oligonucleotide comprising a region that is complimentary to a portion of said flap oligonucleotide;
- and
- d) detecting amplification of one or more different target regions from said bisulfite-treated DNA during PCR-flap assay reactions.
14 more elements
Dependencies are inferred from the claim text.