Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.
The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.
Lichtenstein
A V
AV
Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow 115478, Russia. ali_cht@online.ru
Serdjuk
O I
OI
Sukhova
T I
TI
Melkonyan
H S
HS
Umansky
S R
SR
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D009841:Oligonucleotides; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific