Controlled Ribonucleotide Tailing of cDNA ends (CRTC) by Terminal Deoxynucleotidyl Transferase: A New Approach in PCR-Mediated Analysis of mRNA Sequences
Open Access
- 1 May 1996
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 24 (9) , 1789-1791
- https://doi.org/10.1093/nar/24.9.1789
Abstract
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.Keywords
This publication has 9 references indexed in Scilit:
- Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR.1993
- Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries byin vitroamplificationNucleic Acids Research, 1991
- Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid supportNucleic Acids Research, 1989
- Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.Proceedings of the National Academy of Sciences, 1988
- [5] Terminal transferase: Use in the tailing of DNA and for in Vitro mutagenesisPublished by Elsevier ,1983
- 5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiencyNucleic Acids Research, 1981
- Immunoglobulin light-chain structural gene sequences cloned in a bacterial plasmidNature, 1978
- Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferaseNucleic Acids Research, 1976
- Synthetic PolynucleotidesEuropean Journal of Biochemistry, 1971