A method to increase the sensitivity of mutation specific oligonucleotide hybridization using asymmetric polymerase-chain reaction (PCR).
We propose a simple and reliable method to increase the sensitivity of mutation specific oligonucleotide hybridization (MSOH) at least 2.5 times, when it is used to detect mutations in samples of DNA from tumor tissues. The method is based on using single stranded (ss) DNA, amplified by asymmetric PCR, as a target for MSOH analysis. During the first step, genomic DNA, isolated from tissue samples, has to be amplified by "standard", symmetric PCR, with sense and antisense primers in equimolar concentration. This amplification can be performed in a diminished volume of reaction mixture. In the second step obtained double stranded (ds) PCR DNA-product can be used as a template for asymmetric PCR, using only a single primer. The ss DNA must be complementary to the set of mutation specific oligonucleotides. By this innovation we have been able to clarify questionable results of MSOH using ds DNA as a target. Comparing MSOH from ss DNA to that from ds DNA, the observed rate of Gs-alpha mutations in thyroid tumor tissue samples increased to 16.7% (14/66) from 6% (4/66).
Gorelov
V N
VN
Department of Surgery A, Heinrich-Heine-University, Düsseldorf, Germany.
Röher
H D
HD
Goretzki
P E
PE
D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016376:Oligonucleotides, Antisense; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013964:Thyroid Neoplasms / Q000235:genetics*
D003062:Codon; D017931:DNA Primers; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D016376:Oligonucleotides, Antisense