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US 11,193,168 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.
Claims13
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 said 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 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 (a), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair.
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Dependencies are inferred from the claim text.