Highly sensitive revealing of PCR products with capillary electrophoresis based on single photon detection.
Post-PCR fragment analysis was conducted using our single photon detection-based DNA sequencing instrument in order to substantially enhance the detection of nucleic biomarkers. Telomerase Repeat Amplification Protocol assay was used as a model for real-time PCR-based amplification and detection of DNA. Using TRAPeze XL kit, telomerase-extended DNA fragments were obtained in extracts of serial 10-fold dilutions of telomerase-positive cells, then amplified and detected during 40-cycle real-time PCR. Subsequently, characteristic 6-base DNA ladder patterns were revealed in the post-PCR samples with capillary electrophoresis (CE). In our CE instrument, fluorescently labeled DNA fragments separate in a single-capillary module and are illuminated by a fiberized Ar-ion laser. The laser-induced fluorescence (LIF) is filtered and detected by the fiberized single photon detector (SPD). To assess the sensitivity of our instrument, we performed PCR at fewer cycles (29 and 25), so that the PCR machine could detect amplification only in the most concentrated samples, and then examined samples with CE. Indeed, PCR has detected amplification in samples with minimum 10(4) cells at 29 cycles and over 10(5) cells at 25 cycles. In contrast, the SPD-based CE-LIF has revealed 6-base repeats in samples with as low as 10(2) cells after 29 cycles and 10(3) cells after 25 cycles. Thus, we have demonstrated 100- to 1000-fold increase in the sensitivity of biomarker detection over real-time PCR, making our approach especially suitable for analysis of clinical samples where abundant PCR inhibitors often cause false-negative results.
Kabotyanski
Evgeni A
EA
Department of Electrical and Computer Engineering, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. eak@dnalab.cc
Botchkina
Inna L
IL
Kosobokova
Olga
O
Botchkina
Galina I
GI
Gorfinkel
Vera
V
Gorbovitski
Boris
B
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods; D006801:Humans; D017785:Photons; D016133:Polymerase Chain Reaction / Q000379:methods; D011933:Reagent Kits, Diagnostic
D011933:Reagent Kits, Diagnostic