Abstract
BackgroundMultiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them.ResultsHere, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts) in the new templates.ConclusionFormation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.
Highlights
Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources
The molecular mechanism that leads to the rearrangements was proven by sequencing 475 chimeric junctions generated by MDA
An MDA reaction from a single E. coli cell was analyzed by the 454 Life Sciences pyrosequencing method [12]. 495 chimeras were found in the 108,944 total uniquely mapped reads (10,878,753 total uniquely mapped bases) of E. coli K12 sequence
Summary
Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Multiple displacement amplification (MDA) [1,2] is used to amplify plasmids [1], and BACs [3], and for whole genome amplification [2], for DNA from limiting samples [4], directly from small biological specimens [5], and from single bacterial cells for use in DNA sequencing [6]. The molecular mechanism that leads to the rearrangements was proven by sequencing 475 chimeric junctions generated by MDA
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call