HIGHLY SENSITIVE METHOD FOR DETECTING LOW FREQUENCY MUTATIONS

摘要

The disclosed edge-blocker oligonucleotide based AS-NEPB-PCR method amplifies allele specific DNA (or RNA) while dramatically blocking amplification of wild type (WT) DNA (or RNA). The AS-NEPB-PCR design allows ready modification of an existing PCR reaction setup with an edge-blocker oligonucleotide together with an allele specific primer complementary to the mutant sequence to achieve allele specific amplification. The method simplifies assay optimization procedures and achieved sensitivity sufficient to detect a signal present at 0.1% level with close to 100% specificity, which is useful in detecting SNP or genetic mutations. The method was used to detect three different genetic mutations in cancer, in KRAS, BRAF, and EGFR, with three different types of modified edge-blocker oligonucleotides (phosphate, inverted dT and amino-C7). It was possible to detect one copy of mutant DNA in 1000-copy of normal DNA background of a heterogeneous sample, and was far more sensitive than the other blocking method.

主权项

1. A method for detecting, in the presence of a wild type sequence, a mutant nucleic acid sequence defined by one or more mutation due to at least one or more of a substitution, a deletion or an insertion at a position while suppressing the signal due to the wild type sequence, the method comprising the steps of:
selecting an allele specific primer corresponding to a portion of the mutant nucleic acid sequence such that a 3′ end of the allele specific primer aligns with at least one mutated nucleic acid position without a mismatch; selecting an edge-blocker wild type oligonucleotide corresponding to the wild type sequence such that the 3′ end of the edge-blocker wild type oligonucleotide has at least one mismatch at or about its 3′ end, and, wherein, furthermore maybe similar as CBO method, the 3′ end of the edge-blocker wild type oligonucleotide is blocked whereby making it non-extendable in a polymerase chain reaction; selecting one or more reverse primers; selecting one or more probes to detect amplification products of the polymerase chain reaction; carrying out the polymerase chain reaction with the ingredients comprising the allele specific primer, the edge-blocker wild type oligonucleotide, the one or more reverse primers and the one or more probes.