Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.
Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.
Schmidt
W M
WM
Vienna Biocenter, Institute of Microbiology and Genetics, Austria.
Mueller
M W
MW
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D011088:DNA Ligases / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics*; D006157:Guanosine Monophosphate; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D018076:DNA, Complementary; D012333:RNA, Messenger; D006157:Guanosine Monophosphate; D004253:DNA Nucleotidylexotransferase; D011088:DNA Ligases