High throughput mutation screening methods and kits using a universalized approach - differential sequence fill-in (dsf)-enabled sequential adapter ligation and amplification

摘要: This disclosure teaches high throughput mutation screening methods allowing simultaneous analysis of multiple genetic regions interest and sensitive detection very low frequency mutation(s) by the use a universalized approach. Methods comprise treating RNA:DNA heteroduplexes with ribonuclease, sequence extension an RNA-primed DNA polymerase, ligation blocking adapter, differential fill-in followed single-strand-specific nuclease digestion to permit full-length subsequent tagged reporter adapter solely in mutants filled complementary deoxyribonucleotide triphosphate. By forming mutant-dual hybrids or mutant-triple hybrids, and/or quantification may be directed commonly shared tag(s) flanking sequences for signal detection/enhancement amplification all different regardless source number mutations involved, thereby avoiding tremendous effort target-specific amplifications. performed wholly partially solution, on solid phase media, large scale, adapted automated semi-automated analysis, any combinations thereof.