The use of multiple displacement amplification to amplify complex DNA libraries.
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.
Fullwood
Melissa J
MJ
Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, Genome #02-01, Singapore.
Tan
Jack J S
JJ
Ng
Patrick W P
PW
Chiu
Kuo Ping
KP
Liu
Jun
J
Wei
Chia Lin
CL
Ruan
Yijun
Y
D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D023281:Genomics / Q000379:methods; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000592:standards; D010957:Plasmids / Q000235:genetics; D011786:Quality Control; D016324:Sequence Tagged Sites