HIGH THROUGHPUT MUTATION SCREENING METHODS AND KITS

摘要

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