The use of multiple displacement amplification to amplify complex DNA libraries

Nucleic Acids Res. 2008 Mar;36(5):e32. doi: 10.1093/nar/gkn074. Epub 2008 Feb 19.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Cells, Cultured
  • Escherichia coli / genetics
  • Gene Library*
  • Genetic Vectors
  • Genomics / methods
  • Humans
  • Nucleic Acid Amplification Techniques* / standards
  • Plasmids / genetics
  • Quality Control
  • Sequence Tagged Sites