Enhanced solid phase PCR: mechanisms to increase priming by solid support primers.
Conventional solid phase amplification regimens such as solid phase PCR (SP-PCR), asymmetric SP-PCR, and bridge PCR are mechanistically limited with respect to amplification efficiency and solid support primer involvement. Here we present enhanced solid phase PCR (ESP-PCR) in which solid support primer priming is facilitated by its nesting and high melting temperature (T(m)) relative to the aqueous counterpart. In the study, we demonstrated increased solid support surface loading using ESP-PCR versus standard SP-PCR for three diagnostic targets: Neisseria gonorrhoeaeopa (9.89-fold), N. gonorrhoeae pilS (2.14-fold), and Chlamydia trachomatis cryptic plasmid orf3 (1.41-fold). Furthermore, we applied ESP-PCR to detect five copies of N. gonorrhoeae and C. trachomatis DNA.
Khan
Zaheer
Z
Genera Biosystems, Walter and Eliza Hall Institute Biotechnology Centre, Bundoora, Victoria 3083, Australia.
Poetter
Karl
K
Park
Daniel J
DJ
D002692:Chlamydia trachomatis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D008863:Microspheres; D009344:Neisseria gonorrhoeae / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry
D017931:DNA Primers; D004269:DNA, Bacterial; D012822:Silicon Dioxide