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Mechanism of chimera formation during the Multiple Displacement Amplification reaction

BACKGROUND: 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...

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Autores principales: Lasken, Roger S, Stockwell, Timothy B
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855051/
https://www.ncbi.nlm.nih.gov/pubmed/17430586
http://dx.doi.org/10.1186/1472-6750-7-19
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author Lasken, Roger S
Stockwell, Timothy B
author_facet Lasken, Roger S
Stockwell, Timothy B
author_sort Lasken, Roger S
collection PubMed
description BACKGROUND: 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. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. RESULTS: Here, 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. CONCLUSION: Formation 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.
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spelling pubmed-18550512007-04-24 Mechanism of chimera formation during the Multiple Displacement Amplification reaction Lasken, Roger S Stockwell, Timothy B BMC Biotechnol Methodology Article BACKGROUND: 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. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. RESULTS: Here, 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. CONCLUSION: Formation 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. BioMed Central 2007-04-12 /pmc/articles/PMC1855051/ /pubmed/17430586 http://dx.doi.org/10.1186/1472-6750-7-19 Text en Copyright © 2007 Lasken and Stockwell; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Lasken, Roger S
Stockwell, Timothy B
Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title_full Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title_fullStr Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title_full_unstemmed Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title_short Mechanism of chimera formation during the Multiple Displacement Amplification reaction
title_sort mechanism of chimera formation during the multiple displacement amplification reaction
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855051/
https://www.ncbi.nlm.nih.gov/pubmed/17430586
http://dx.doi.org/10.1186/1472-6750-7-19
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