Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance.
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.
Lareu
Ricky R
RR
Tissue Modulation Laboratory, Division of Bioengineering, Faculty of Engineering, National University of Singapore, Division Office Block E3A #04-15, 7 Engineering Drive 1, Singapore 117574, Singapore.
Harve
Karthik S
KS
Raghunath
Michael
M
D018929:Cell Culture Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D007424:Intracellular Fluid / Q000737:chemistry*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004247:DNA; D004259:DNA-Directed DNA Polymerase