CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.
Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs.
Schmidt
W M
WM
VBC-GENOMICS Research GmbH, PO Box 207, A-1091 Vienna, Austria.
Mueller
M W
MW
D002460:Cell Line; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012315:RNA Caps; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D013698:Templates, Genetic
D018076:DNA, Complementary; D012315:RNA Caps; D012333:RNA, Messenger; D008345:Manganese; D004253:DNA Nucleotidylexotransferase; D012194:RNA-Directed DNA Polymerase