Random mutagenesis-PCR to introduce alterations into defined DNA sequences for 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 mutations. One problem, common to all methods of SNP and mutation detection, is that experimental conditions required for detection of DNA sequence variants depend on the specific DNA sequence to be analyzed. Although algorithms and other calculations have been developed to predict the experimental conditions required to detect DNA sequence variation in a specific DNA sequence, these algorithms do not always provide reliable information and experimental conditions for SNP and mutation detection must be devised empirically. Determination of experimental conditions for detection of DNA sequence variation is difficult when samples containing only wild type sequence are available. When patient derived positive controls are used, increasingly there are valid concerns about commercial ownership and patient privacy. This report presents a rapid and efficient method, employing random mutagenesis-PCR (RM-PCR) using low fidelity DNA polymerase, to randomly introduce single and multiple base substitutions and deletions into DNA sequences of interest. Clones with sequence changes were used to validate denaturing HPLC (DHPLC) algorithm predictions, optimize conditions for mutation detection in exon 15 of the tyrosine kinase domain of the MET proto-oncogene, and to confirm the association between specific DNA sequence changes and unique DHPLC chromatographic 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 discussed along with challenges associated with detecting sequence alterations in mixed tumor/normal tissue, pooled samples, and from regions of the genome that have been amplified during tumorigenesis or duplicated during evolution. Hum Mutat 17:210-219, 2001. Published 2001 Wiley-Liss, Inc.
Nickerson
M L
ML
Laboratory of Immunobiology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, USA. nickersm@mail.ncifcrf.gov
Warren
M B
MB
Zbar
B
B
Schmidt
L S
LS
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005091:Exons / Q000235:genetics; D006801:Humans; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics; D017422:Sequence Analysis, DNA
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D004247:DNA; D019859:Proto-Oncogene Proteins c-met