Please use this identifier to cite or link to this item:
Title: Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence
Authors: Harve, K.S. 
Lareu, R.
Rajagopalan, R. 
Raghunath, M. 
Issue Date: 23-Oct-2009
Source: Harve, 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.
Abstract: Amplification 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.
Source Title: Nucleic Acids Research
ISSN: 03051048
DOI: nar/gkp884
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
2009-understanding_how_crowded_interior_cells-published.pdf2.8 MBAdobe PDF




checked on Feb 12, 2018

Page view(s)

checked on Mar 12, 2018


checked on Mar 12, 2018

Google ScholarTM



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.