Please use this identifier to cite or link to this item: https://doi.org/10.1093/nar/gkp884
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dc.titleUnderstanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence
dc.contributor.authorHarve, K.S.
dc.contributor.authorLareu, R.
dc.contributor.authorRajagopalan, R.
dc.contributor.authorRaghunath, M.
dc.date.accessioned2014-04-25T09:05:38Z
dc.date.available2014-04-25T09:05:38Z
dc.date.issued2009-10-23
dc.identifier.citationHarve, K.S., Lareu, R., Rajagopalan, R., Raghunath, M. (2009-10-23). Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence. Nucleic Acids Research 38 (1) : 172-181. ScholarBank@NUS Repository. https://doi.org/10.1093/nar/gkp884
dc.identifier.issn03051048
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/51826
dc.description.abstractAmplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA-DNA and DNA-RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA-single-strands. © The Author(s) 2009. Published by Oxford University Press.
dc.sourceScopus
dc.typeArticle
dc.contributor.departmentANATOMY
dc.contributor.departmentCHEMICAL & BIOMOLECULAR ENGINEERING
dc.contributor.departmentBIOENGINEERING
dc.description.doi10.1093/nar/gkp884
dc.description.sourcetitleNucleic Acids Research
dc.description.volume38
dc.description.issue1
dc.description.page172-181
dc.description.codenNARHA
dc.identifier.isiut000273229100020
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