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 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.