Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

摘要

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.