Patent ID: 7947446

Claim:
A method of simultaneously screening for genetic mutations in different genes in a multitude of samples, the method comprising: providing a sample of heteroduplex molecules, wherein the heteroduplex molecules each include: one strand of a ribonucleic acid (RNA) to be tested; and one strand of an antisense deoxyribonucleic acid (DNA) probe; wherein the sample forms: a first population of fully hybridized wild-type heteroduplexes; a second population of mutant heteroduplexes having a single unhybridized ribonucleotide; and a third population of homologous heteroduplexes having at least one stretch of two or more unhybridized ribonucleotides; incubating the sample of heteroduplex molecules with a ribonuclease enzyme, wherein the ribonuclease enzyme cleaves the unhybridized ribonucleotides in the second population and the third population and exposes a 3′ hydroxyl group at the cleavage sites; protecting the 3′ end of the RNA strand; performing differential sequence fill-in at the cleaved sites using an RNA-primed DNA polymerase to create a second sample; and incubating the second sample with a single-strand-specific nuclease to digest the unprotected deoxyribonucleotides in the third population and in the second population not filled in with a complementary dNTP; wherein the first population and heteroduplexes in the second population filled in with a complementary dNTP are undigested; incubating the undigested portion of the second sample with: a DNA-dependent DNA polymerase; and all four dNTPs; wherein the DNA-dependent DNA polymerase creates: a full-length sequence extension in the second population; and a new 3′ single nucleotide overhang carrying a free 3′-hydroxyl group that is unprotected; linking a tagged reporter adapter to the unprotected overhang to form marked mutant-adapter hybrids; and detecting said marked mutant-adapter hybrids.