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Title: Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance
Authors: Lareu, R.R.
Harve, K.S. 
Raghunath, M. 
Keywords: DNA polymerase
Excluded volume effect
Macromolecular crowding
Polymerase chain reaction
Reverse transcriptase
Reverse transcription
Issue Date: 9-Nov-2007
Source: Lareu, R.R., Harve, K.S., Raghunath, M. (2007-11-09). Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance. Biochemical and Biophysical Research Communications 363 (1) : 171-177. ScholarBank@NUS Repository.
Abstract: The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding. © 2007 Elsevier Inc. All rights reserved.
Source Title: Biochemical and Biophysical Research Communications
ISSN: 0006291X
DOI: 10.1016/j.bbrc.2007.08.156
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