Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs.
A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Spain.
B
B
Navarro
J A
JA
Daròs
R
R
Flores
D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D004256:DNA Polymerase I; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D017403:In Situ Hybridization; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification; D000079962:RNA, Circular; D019244:RNA, Satellite / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D017422:Sequence Analysis, DNA; D014772:Viroids / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D000079962:RNA, Circular; D019244:RNA, Satellite; D012367:RNA, Viral; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I