A genotypic mutation system measuring mutations in restriction recognition sequences

Abstract

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the Pvull recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'Pvull mutant standard'. Following amplification with Taqpolymerase and cloning into λgt10, plaques containing wild-type sequence, Pvull mutant standard or Taqpolymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 Pvull mutant standard copies can be rescued from 108 to 109 wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the Mspl-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 108 copies of wild-type DNA. © 1991 Oxford University Press.