DNA Methyltransferase Genotyping (DMG) is a patented new technique in which sequence specific MTases are used to rapidly fingerprint DNA. The presence or absence of MTase recognition sites is determined using a 3-step procedure: (1) DNA is PCR- amplified.(2) Amplified DNA is enzymatically methylated at specific 2 to 6 b.p. sites. (3) Methylated DNA is detected radiometrically or immunochemically. Since gel electrophoresis is not required, DMC is extremely fast. Using a convenient Scintillation Proximity Assay (SPA) to detect 3H-methyl-DNA, a viral infectious disease (HSV-2) and a heritable human disease (MFAA allele of Neonatal Alloimmune Thrombocytopenia) can be diagnosed in 20-30 minutes. If biotinylated primers and combinations of restriction enzymes and MTases are used, then ordered maps of amplicons can be constructed by Streptavidin-SPA bead capture in a few minutes. Phase II Research is proposed to further develop DMG for two medical diagnostic applications (one heritable and one infectious disease). In an automated format, it should be possible to diagnose many diseases in <15 minutes. Additional """"""""Long PCR"""""""" experiments are proposed to construct 1-10 kb ordered maps; and to immunochemically """"""""paint"""""""" DNA using MTases and monoclonal antibodies to methylated bases.
Medical diagnostic products include automated genotyping instrument, MTases and diagnostic kits. Research products include R-M enzymes and reagents for ordered mapping. Immunochemical products include """"""""DNA paint"""""""" reagents: MTases, Antibodies to methylated bases, and matched immunostaining reagents (200 ways to """"""""paint"""""""" DNA).
| Lopez, Osvaldo J; Quintanar, Andre; Padhye, Nisha V et al. (2003) Genotyping of DNA using sequence-specific methyltransferases followed by immunochemical detection. J Immunoassay Immunochem 24:11-28 |
