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A sensitive method for detection of mutations--a PCR-based RNase protection assay
K K Murthy1, S H Shen, D Banville
1Pharmaceutical Biotechnology Sector, NRCC, Montreal, Quebec, Canada.
DNA and Cell Biology
|January 1, 1995
Summary
A novel RNase protection assay effectively detects DNA point mutations in large gene segments. This sensitive method identifies single-base changes, insertions, and deletions, improving upon existing techniques.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Current DNA mutation detection methods have limitations, including hazardous chemical use or inability to analyze large DNA fragments.
- Existing techniques like chemical mismatch cleavage, single-stranded conformational polymorphism, and denaturing gradient gel electrophoresis are often restricted in sensitivity or fragment size.
- There is a need for a reliable and sensitive method to detect mutations across extensive DNA regions.
Purpose of the Study:
- To develop a novel, sensitive, and reliable RNase protection assay for detecting point mutations in large DNA segments.
- To overcome the limitations of existing mutation detection technologies.
Main Methods:
- Developed a novel RNase protection assay utilizing RNase I.
- Amplified target DNA using the polymerase chain reaction (PCR).
- Hybridized amplified DNA with a 32P-labeled wild-type RNA probe, followed by RNase I digestion to cleave mismatches. Separated and detected resulting RNA fragments using denaturing polyacrylamide-urea gel electrophoresis and autoradiography.
Main Results:
- The assay efficiently detected single-base changes (all four base substitutions) at mismatch sites.
- Demonstrated the method's capability to detect insertions and single-base deletions.
- Successfully identified a single-base deletion in PTP1C (motheaten mutation) in mice using PCR-amplified genomic DNA, distinguishing heterozygotes from wild-type and homozygotes.
Conclusions:
- The developed PCR-based RNase protection assay is a sensitive and reliable method for detecting various DNA mutations, including point mutations, insertions, and deletions, in large DNA fragments.
- This technique offers an improvement over existing methods, particularly for analyzing extensive genomic regions.
- The assay has practical applications in genetic studies, as shown by its ability to genotype mice for the motheaten mutation.