CONTROLLED RIBONUCLEOTIDE TAILING OF CDNA ENDS (CRTC) BY TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE - A NEW APPROACH IN PCR-MEDIATED ANALYSIS OFMESSENGER-RNA SEQUENCES

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deox ynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequenc e 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 a nchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DN A ligase-dependent ligation. PCR amplification, mediated by two sequen ce-specific primers, yields the desired unique product suitable for cl oning and dideoxy-sequencing.