Patent ID: 7790393

Claim:
A method of multiplex amplification of nucleic acid target regions, comprising: a) providing a sample containing genomic DNA, wherein said genomic DNA comprises a plurality nucleic acid target regions, wherein each nucleic acid target region comprises a footprint region that is at least twenty bases in length and is suspected of containing a SNP; b) amplifying said plurality of nucleic acid target regions from said genomic DNA to produce a first set of amplified products comprising amplified nucleic acid target regions, wherein said amplifying is in a first polymerase chain reaction mixture comprising a plurality of primer pairs, wherein each primer in said plurality of primer pairs in said first polymerase chain reaction mixture is present at essentially the same initial molar concentration, and wherein said primer pairs are each configured to amplify a nucleic acid target region; c) determining an amplification factor F for each amplified nucleic acid target region in said first set of amplified products, wherein said amplification factor F is the ratio of amplified nucleic acid target region concentration after amplification to initial nucleic acid target region concentration, d) determining an apparent initial primer concentration from said determined amplification factor F for each nucleic acid target region, wherein: F =(2− e −k a ct ) n wherein k a is the association rate constant of primer annealing, c is the initial primer concentration, t is the primer annealing time and n is the number of PCR cycles, e) determining a relative primer concentration value R[n] for each given nucleic acid target region, wherein R[n] is equal to the highest observed apparent primer concentration of all amplified nucleic acid target regions in said first set of amplified products, divided by the apparent primer concentration for the given amplified nucleic acid target region; f) determining a normalized primer concentration, wherein the normalized primer concentration for each given nucleic target region, is the value of R[n] for the corresponding amplified nucleic acid target region of step e) multiplied by the initial molar concentration of primers used in step b); g) amplifying said plurality of nucleic acid target regions from said genomic DNA to produce a second set of amplified products, wherein said amplifying is in a second polymerase chain reaction mixture comprising a plurality of primer pairs, wherein said primers in said plurality of primer pairs in said second polymerase chain reaction mixture are present in said normalized primer concentrations so as to balance the amplification factors for said amplified nucleic acid target regions in said second set of amplified products.