A novel blocker-PCR method for detection of rare mutant alleles in the presence of an 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 1:10(3). 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 10(4) times excess amount of normal DNA.
Seyama
T
T
Department of Radiobiology, Radiation Effects Research Foundation, Hiroshima, Japan.
Ito
T
T
Hayashi
T
T
Mizuno
T
T
Nakamura
N
N
Akiyama
M
M
D000483:Alleles; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics; D012680:Sensitivity and Specificity; D013696:Temperature
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides