Technical Report No. 17-92
A novel blocker-PCR method for detection of rare mutant alleles in the presence of an excess amount of normal DNA
Seyama T, Ito T, Hayashi T, Mizuno T, Nakamura N, Akiyama MNucleic Acids Res 20(10):2493-6, 1992
Summary
A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:103. Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 104 times excess amount of normal DNA.
A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:103. Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 104 times excess amount of normal DNA.