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dc.contributor.authorLareu, Ricky R.
dc.contributor.authorHarve, K.
dc.contributor.authorRaghunath, M.
dc.date.accessioned2017-01-30T14:56:40Z
dc.date.available2017-01-30T14:56:40Z
dc.date.created2014-02-25T20:00:39Z
dc.date.issued2007
dc.identifier.citationLareu, Ricky R. and Harve, Karthik S. and Raghunath, Michael. 2007. Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance. Biochemical and Biophysical Research Communications. 363 (1): pp. 171-177.
dc.identifier.urihttp://hdl.handle.net/20.500.11937/41966
dc.identifier.doi10.1016/j.bbrc.2007.08.156
dc.description.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.

dc.publisherAcademic Press
dc.subjectSensitivity
dc.subjectMacromolecular crowding
dc.subjectReverse transcriptase
dc.subjectMacromolecule
dc.subjectPolymerase chain reaction
dc.subjectDNA polymerase
dc.subjectExcluded volume effect
dc.subjectReverse transcription
dc.titleEmulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance
dc.typeJournal Article
dcterms.source.volume363
dcterms.source.startPage171
dcterms.source.endPage177
dcterms.source.issn0006-291X
dcterms.source.titleBiochemical and Biophysical Research Communications
curtin.department
curtin.accessStatusFulltext not available


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