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CapSelect: A highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs

1999; Oxford University Press; Volume: 27; Issue: 21 Linguagem: Inglês

10.1093/nar/27.21.e31-i

1362-4962

Wolfgang M. Schmidt, Manfred W. Mueller,

Advanced biosensing and bioanalysis techniques

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.