A sensitive method for detection of mutations--a PCR-based RNase protection assay.
Several techniques are currently available for detecting point mutations in DNA. The most widely used methods either use hazardous chemicals (chemical mismatch cleavage) or can detect mutations only in short (200- to 500-bp) fragments (single-stranded conformational polymorphism and denaturing gradient gel electrophoresis). In an effort to develop a sensitive and reliable method for the detection of mutations in large segments of DNA, a novel RNase protection assay using RNase I was developed. In this method, the desired portion of the gene is amplified by the polymerase chain reaction (PCR) using specific oligonucleotides and hybridized to a 32P-labeled RNA probe containing the wild-type sequence. The RNA/DNA hybrid is subsequently digested with RNase I, which cleaves the RNA at the mismatch sites. The protected RNA fragments are separated on a denaturing polyacrylamide-urea gel and detected by autoradiography. Four different RNA probes from two protein tyrosine phosphatases (PTP1C and PTP2C) were assayed using this procedure. Several mutants of the two enzymes were tested using wild-type RNA probes. Single-base changes involving all four bases at the mismatch site could be detected efficiently. The ability of this method to detect insertions and single-base deletions was also demonstrated. Using a PCR-based RNase protection assay, a single-base deletion in PTP1C in the motheaten mutation in mice could be detected. Using fragments amplified from genomic DNA, mice that were heterozygous for the motheaten mutation could be distinguished from wild type and homozygotes for this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
Pharmaceutical Biotechnology Sector, NRCC, Montreal, Quebec, Canada.
K K
KK
Murthy
S H
SH
Shen
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D
Banville
D000818:Animals; D004252:DNA Mutational Analysis; D006580:Genetic Carrier Screening / Q000379:methods; D005821:Genetic Techniques; D051379:Mice; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D015347:RNA Probes; D012259:Ribonuclease, Pancreatic; D012680:Sensitivity and Specificity
D009692:Nucleic Acid Heteroduplexes; D015347:RNA Probes; D012259:Ribonuclease, Pancreatic; D017027:Protein Tyrosine Phosphatases