A highly efficient method for long-chain cDNA synthesis using trehalose and betaine

被引：50

作者：

Spiess, AN ^{[1
]}

Ivell, R ^{[1
]}

机构：

[1] Inst Hormone & Fertil Res, D-22529 Hamburg, Germany

来源：

ANALYTICAL BIOCHEMISTRY | 2002年 / 301卷 / 02期

关键词：

cDNA synthesis; secondary structure; melting temperature; thermostabilization;

D O I：

10.1006/abio.2001.5474

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2002 Elsevier Science (USA).

引用

页码：168 / 174

页数：7

共 22 条

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