Random mutagenesis-PCR to introduce alterations into defined DNA sequencesfor validation of SNP and mutation detection methods

Sensitive and high throughput techniques are required for the detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs) and mu tations. One problem, common to all methods of SNP and mutation detection, is that experimental conditions required for detection of DNA sequence vari ants depend on the specific DNA sequence to be analyzed. Although algorithm s and other calculations have been developed to predict the experimental co nditions required to detect DNA sequence variation in a specific DNA sequen ce, these algorithms do not always provide reliable information and experim ental conditions for SNP and mutation detection must be devised empirically . Determination of experimental conditions for detection of DNA sequence va riation is difficult when samples containing only wild type sequence are av ailable. When patient derived positive controls are used, increasingly ther e are valid concerns about commercial ownership and patient privacy. This r eport presents a rapid and efficient method, employing random mutagenesis-P CR (RM-PCR) using low fidelity DNA polymerase, to randomly introduce single and multiple base substitutions and deletions into DNA sequences of intere st. Clones with sequence changes were used to validate denaturing HPLC (DHP LC) algorithm predictions, optimize conditions for mutation detection in ex on 15 of the tyrosine kinase domain of the MET proto-oncogene, and to confi rm the association between specific DNA sequence changes and unique DHPLC c hromatographic profiles (signatures). Finally, DNA from 33 papillary renal carcinoma (PRC) patients was screened for mutations in exon 15 of MET using "validated" DHPLC conditions as a proof of principle application of RM-PCR . Use of RM PCR for DHPLC and other SNP/mutation detection methods is discu ssed along with challenges associated with detecting sequence alterations i n mixed tumor/normal tissue, pooled samples, and from regions of the genome that have been amplified during tumorigenesis or duplicated during evoluti on. Hum Mutat 17:210-219, 2001. Published 2001 Wiley-Liss, Inc.(dagger).