Methods and compositions to enable multiplex cold-pcr

Abstract

The present invention is directed to methods, compositions and reaction mixtures for multiplexing COLD-PCR/ice-COLD-PCR to enrich simultaneously several low abundance alleles (mutant target sequences) from a sample. The invention also involves COLD-PCR/ice-COLD-PCR amplification performed on DNA fragments that have different melting temperatures, and therefore different critical denaturation temperatures, in a graded temperature approach such that mutation enrichment is achieved on all diverse DNA fragments simultaneously (temperature-independent COLD-PCR or TI-COLD-PCR). The invention also involves methods for enabling identification of variant-sequence alleles in the presence of a large excess of non-variant alleles in nucleic acids without the complication of polymerase-introduced errors or other primer-introduced artifacts.