Methods for identifying multiple DNA alteration markers in a large background of wild-type DNA
AssigneeCleveland State University
InventorBaochuan Guo
Life of this patent
Estimating the term…
Abstract
Methods for simultaneously surveying the status of a large number of DNA mutation markers are described. In addition, methods for simultaneously determining the methylation status at multiple sites of a collection of genes, in a single assay, are described.
Claims43
A method for surveying the status of multiple mutation markers in a large background of wild-type DNA, the method comprising:
- providing a sample including mutant DNA and wild-type DNA, the mutant DNA including mutations;
- amplifying the sample by a first PCR to generate DNA fragments containing the mutation sites;
- wherein the first PCR is multiplexed PCR utilizing two or more pairs of PCR primers, wherein each pair of primers includes a forward primer and a reverse primer, wherein each pair of primers is adapted to amplify a sequence containing one or more of the mutation sites and wherein each primer includes both a target-specific part and a universal tail part, wherein the target-specific part flanks the sequence to allow for amplification of the sequence and the sequence of the universal tail part is the same for all forward and reverse primers, wherein the universal tail cannot bind to human genomic sequence;
- enriching the mutant DNA fragments containing the mutations from the amplicons of the multiplexed PCR simultaneously by performing one or multiple mutant-specific enrichment cycles including competing mutation-specific hybridization and extraction to thereby form an enriched system;
- amplifying the enriched system by a second PCR to generate sufficient amounts of mutant DNA for detection;
- and
- simultaneously surveying the status of the target mutation sites.
A method for producing sufficiently pure mutant DNA fragments for determining the mutation status at a plurality of DNA mutation sites in a large background of wild-type DNA, the method comprising:
- providing a DNA sample including both mutant DNA and wild-type DNA;
- amplifying DNA sequences including the mutation sites by multiplexed PCR to thereby produce amplicons;
- and enriching mutant DNA fragments having the mutations from the amplicons of the PCR simultaneously by competing mutation-specific hybridization and extraction;
- wherein the multiplexed PCR utilizes two or more pairs of PCR primers, wherein each pair of the primers hybridize to a sequence containing one or more of the mutation sites for amplification of the sequence.
A method for surveying the status of multiple mutation markers in a large background of wild-type DNA, the method comprising:
- (a) providing a sample including mutant DNA and wild-type DNA, the mutant DNA including mutations;
- (b) amplifying the sample from step (a) by a first PCR to generate DNA fragments containing the mutation sites;
- (c) enriching the mutant DNA fragments from step (b) containing the mutations by performing one or multiple mutant-specific enrichment cycles to thereby form an enriched system;
- (d) amplifying the enriched system from step (c) by a second PCR to generate sufficient amounts of mutant DNA for detection;
- and
- (e) surveying the status of the target mutation sites after completing the amplifying step (d).
Dependencies are inferred from the claim text.