A Highly Efficient Method for Long-Chain cDNA Synthesis Using Trehalose and Betaine

✍ Scribed by Andrej-Nikolai Spiess; Richard Ivell

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 129 KB
• Volume: 301
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5474

✦ Synopsis

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 clathhrin 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.