A highly efficient method for long-chain cDNA synthesis using trehalose and betaine.
Obtaining full-length cDNA is important for many molecular biology methods like cDNA library construction or RACE-PCR (rapid amplification of cDNA ends). We have found that the inclusion of betaine alone and in combination with trehalose in reverse transcription results in longer cDNA synthesis products. As shown on the 14-kb long clarithrin mRNA with real-time PCR, a combination of 2 M betaine and 0.6 M trehalose leads to almost 9 times more cDNA with a length of 12.5 kb. This is due to the ability of betaine to resolve secondary structures of the RNA, thereby decreasing its melting temperature. The application of betaine in combination with trehalose should prove useful in all laboratory methods relying on full-length cDNA. (USA).
Spiess
Andrej-Nikolai
AN
Institute for Hormone and Fertility Research, Grandweg 64, Hamburg, 22529, Germany. spiess@ihf.de
Ivell
Richard
R
D001622:Betaine / Q000494:pharmacology*; D002966:Clathrin / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis* / Q000235:genetics; D004338:Drug Combinations; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014199:Trehalose / Q000494:pharmacology*
D002966:Clathrin; D018076:DNA, Complementary; D004338:Drug Combinations; D012333:RNA, Messenger; D001622:Betaine; D014199:Trehalose