. Gene mapping and DNA fingerprinting are normally carried out using restriction enzymes. However, for every restriction endonuclease there is a companion methyltransferase (MTase) which has the same DNA sequence specificity. It is therefore possible to use MTases rather than endonucleases to detect mutations and polymorphic sites in DNA. In DNA- MTases genotyping, amplified nucleic acids are methylated using one or more sequence-specific MTases. The presence or absence of DNA MTase recognition sites can be distinguished using 3^H-methyl-SAM as a methyl donor. If a MTase site is present in amplified DNA, then 3^H - methyl groups are incorporated; if no sites are present, then radiolabel is not incorporated. When several different MTase reactions are carried out the pattern of methyl group incorporation defines a DNA MTase genotype. DNA MTase genotyping offers several advantages over restriction (RFLP) mapping and other methods. It is extremely fast, requires no gel electrophoresis, and relies on a simple, quantitative assay. It is also possible to detect methylated DNA immunochemically, so that radioisotope is not required. Phase I Research is proposed to develop DNA MTase genotyping as a rapid (approximately l hour) general purpose genetic fingerprinting technique to identify viral pathogens and mutations responsible for heritable human diseases.
NIH ExPorter
NIH ExPorter