Vn. Gorelov et al., A METHOD TO INCREASE THE SENSITIVITY OF MUTATION SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION USING ASYMMETRIC POLYMERASE CHAIN-REACTION (PCR), Biochemical and biophysical research communications, 200(1), 1994, pp. 365-369
We propose a simple and reliable method to increase the sensitivity of
mutation specific oligonucleotide hybridization (MSOH) at least 2.5 t
imes, when it is used to detect mutations in samples of DNA from tumor
tissues. The method is based on using single stranded (ss) DNA, ampli
fied by asymmetric PCR, as a target for MSOH analysis. During the firs
t step, genomic DNA, isolated from tissue samples, has to be amplified
by ''standard'', symmetric PCR, with sense and antisense primers in e
quimolar concentration. This amplification can be performed in a dimin
ished volume of reaction mixture. In the second step obtained double s
tranded (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 t
he set of mutation specific oligonucleotides. By this innovation we ha
ve 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 t
o 16.7% (14/66) from 6% (4/66). (C) 1994 Academic Press, Inc.