Selective detection of individual DNA molecules by capillary polymerase chain reaction.
On-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection was successfully demonstrated for individual DNA molecules. A single 30-microm-i.d. fused-silica capillary was used both as the reaction vessel and for isolating single molecules. SYBR green I dye was added into the reaction mixture for dynamic fluorescent labeling. Because of the small inside diameter of the capillary, PCR-amplified DNA fragments from single molecules were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. By counting the number of peaks in the capillary via electromigration past a detection window, the number of starting DNA molecules could be determined. With selective primer design, only the molecule of interest was detected. Amplification of the 110-bp fragment from an individual human beta-globin gene and the 142-bp fragment from an individual HIV-1 DNA was demonstrated. This opens the possibility of highly selective and sensitive disease diagnosis at a very early stage.
Li
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Ames Laboratory, US Department of Energy, Iowa 50011, USA.
Xue
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Yeung
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D052160:Benzothiazoles; D001583:Benzoxazoles; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D003959:Diamines; D005456:Fluorescent Dyes; D015683:Gene Products, gag / Q000235:genetics; D005914:Globins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007364:Intercalating Agents; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D011806:Quinolinium Compounds
D052160:Benzothiazoles; D001583:Benzoxazoles; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D015683:Gene Products, gag; D007364:Intercalating Agents; D009930:Organic Chemicals; C099476:PicoGreen; D011804:Quinolines; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; C098022:SYBR Green I; D005914:Globins; D004247:DNA