Fullwood Melissa J, Tan Jack J S, Ng Patrick W P, Chiu Kuo Ping, Liu Jun, Wei Chia Lin, Ruan Yijun
Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Genome #02-01, Singapore.
Nucleic Acids Res. 2008 Mar;36(5):e32. doi: 10.1093/nar/gkn074. Epub 2008 Feb 19.
Complex libraries for genomic DNA and cDNA sequencing analyses are typically amplified using bacterial propagation. To reduce biases, large numbers of colonies are plated and scraped from solid-surface agar. This process is time consuming, tedious and limits scaling up. At the same time, multiple displacement amplification (MDA) has been recently developed as a method for in vitro amplification of DNA. However, MDA has no selection function for the removal of ligation multimers. We developed a novel method of briefly introducing ligation reactions into bacteria to select single insert DNA clones followed by MDA to amplify. We applied these methods to a Gene Identification Signatures with Paired-End diTags (GIS-PET) library, which is a complex transcriptome library created by pairing short tags from the 5' and 3' ends of cDNA fragments together, and demonstrated that this selection and amplification strategy is unbiased and efficient.
用于基因组DNA和cDNA测序分析的复杂文库通常通过细菌繁殖进行扩增。为减少偏差，大量菌落被接种到固体表面琼脂上并刮下。这个过程耗时、繁琐且限制了规模扩大。同时，多重置换扩增（MDA）最近已被开发为一种体外DNA扩增方法。然而，MDA对于去除连接多聚体没有选择功能。我们开发了一种新方法，即短暂地将连接反应引入细菌中以选择单插入DNA克隆，然后进行MDA扩增。我们将这些方法应用于具有双端标签的基因识别签名（GIS-PET）文库，该文库是通过将来自cDNA片段5'和3'末端的短标签配对在一起而创建的复杂转录组文库，并证明这种选择和扩增策略是无偏差且高效的。
