Dilute-'N'-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays.

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dc.contributor.author Jia, Yanwei
dc.contributor.author Osborne, Adam
dc.contributor.author Rice, John E
dc.contributor.author Wangh, Lawrence J
dc.date.accessioned 2019-01-17T19:30:55Z
dc.date.available 2019-01-17T19:30:55Z
dc.date.issued 2010
dc.identifier.issn 1362-4962
dc.identifier.other PMC2887968
dc.identifier.uri https://hdl.handle.net/10192/36118
dc.description.abstract We have recently described a Dilute-'N'-Go protocol that greatly simplifies preparation and sequencing of both strands of an amplicon generated using linear-after-the-exponential (LATE)-PCR, an advanced form of asymmetric PCR . The same protocol can also be used to sequence all limiting primer strands in a multiplex LATE-PCR, by adding back each of the depleted limiting primers to a separate aliquot of the multiplex reaction. But, Dilute-'N'-Go sequencing cannot be used directly to sequence each of the excess primer strands in the same multiplex reaction, because all of the excess primers are still present at high concentration. This report demonstrates for the first time that it is possible to sequence each of the excess primer strands using a modified Dilute-'N'-Go protocol in which blockers are added to prevent all but one of the excess primers serving as the sequencing primer in separate aliquots. The optimal melting temperatures, positions and concentrations of blockers relative to their corresponding excess primers are defined in detail. We are using these technologies to measure DNA sequence changes in mitochondrial genomes that accompany aging and exposure to certain drugs.
dc.format.extent 1 file
dc.language English
dc.language.iso eng
dc.publisher Oxford University Press
dc.relation.isversionof https://dx.doi.org/10.1093/nar/gkq111
dc.rights Creative Commons Attribution-NonCommercial 2.0 UK: England & Wales
dc.rights.uri http://creativecommons.org/licenses/by-nc/2.0/uk/
dc.subject DNA Primers
dc.subject DNA, Mitochondrial
dc.subject Dideoxynucleotides
dc.subject Humans
dc.subject Nucleic Acid Conformation
dc.subject Nucleic Acid Denaturation
dc.subject Nucleic Acid Hybridization
dc.subject Polymerase Chain Reaction
dc.subject Sequence Analysis, DNA
dc.subject DNA Primers
dc.subject DNA, Mitochondrial
dc.subject Dideoxynucleotides
dc.title Dilute-'N'-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays.
dc.type Article
dc.contributor.department Department of Biology
dc.relation.journal Nucleic Acids Research
dc.identifier.pmid 20189962


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Creative Commons Attribution-NonCommercial 2.0 UK: England & Wales Except where otherwise noted, this item's license is described as Creative Commons Attribution-NonCommercial 2.0 UK: England & Wales

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