Selective control of primer usage in multiplex one-step reverse transcription PCR.
BACKGROUND
Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.


RESULTS
Herein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.


CONCLUSIONS
The need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.
Hidalgo Ashrafi
Elena
E
Department of Research and Development, TriLink BioTechnologies, Inc, 9955 Mesa Rim Road, San Diego, CA 92121, USA. ehidalgo@trilinkbiotech.com
Yee
Joyclyn
J
Paul
Natasha
N
D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics; D001923:Brain Chemistry; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013950:Thymus Gland / Q000737:chemistry; D014132:Trachea / Q000737:chemistry
D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase