Selective genotyping of individual cells by capillary polymerase chain reaction.
On-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection was successfully demonstrated for individual human cells. A single 50 num inner diameter (ID) fused-silica capillary served both as the reaction vessel and for isolating single cells. SYBR Green I dye was added into the reaction mixture for dynamic fluorescence labeling. Because of the small ID of the capillary, PCR-amplified DNA fragments from single cells were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. With selective primer design, only cells containing the DNA of interest were amplified. By counting the number of peaks in the capillary via electromigration past a detection window, the number of targeted cell templates could be determined. Identification of the 295 bp fragment beta-actin gene from individual human lymphoblast cell was demonstrated. Independent on-column cell counting provided positive correlation between the starting cell templates and the final PCR products. This opens up the possibility of highly selective and sensitive disease diagnosis at an early stage, when only a few cells in the population are defective.
Li
Hanlin
H
Ames Laboratory-USDOE and Department of Chemistry Iowa State University, Ames, IA 50011, USA.
Yeung
Edward S
ES
D000199:Actins / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D003952:Diagnostic Imaging; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D007834:Lasers; D018655:Lymphocyte Count; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*
D000199:Actins; D004247:DNA