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Meta-PCR: A Novel Method for Creating Chimeric DNA Molecules and Increasing the Productivity of Mutation Scanning Techniques
- Source :
- Genetic Testing. 3:173-183
- Publication Year :
- 1999
- Publisher :
- Mary Ann Liebert Inc, 1999.
-
Abstract
- Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.
- Subjects :
- DNA Repair
Base Pair Mismatch
DNA repair
DNA Mutational Analysis
Molecular Sequence Data
Mutant
Biology
Polymerase Chain Reaction
chemistry.chemical_compound
Exon
Genes, Neurofibromatosis 2
Humans
Gene
Genetics (clinical)
Adaptor Proteins, Signal Transducing
DNA Primers
Genetics
Base Sequence
Chimera
Point mutation
Nuclear Proteins
DNA
Exons
Neoplasm Proteins
chemistry
Evaluation Studies as Topic
DNA mismatch repair
Primer (molecular biology)
Carrier Proteins
MutL Protein Homolog 1
Subjects
Details
- ISSN :
- 15577473 and 10906576
- Volume :
- 3
- Database :
- OpenAIRE
- Journal :
- Genetic Testing
- Accession number :
- edsair.doi.dedup.....2df3e24173477a7615475101fa0b49f6