Nanoliter scale PCR with TaqMan detection.
We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.
Kalinina
O
O
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda MD 20892, USA.
Lebedeva
I
I
Brown
J
J
Silver
J
J
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D005456:Fluorescent Dyes; D006238:Haploidy; D006801:Humans; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011336:Probability; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D000199:Actins; D017931:DNA Primers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D015345:Oligonucleotide Probes; D011933:Reagent Kits, Diagnostic; D004247:DNA