Simplified procedures for detection of amplified DNA using fluorescent label incorporation and reverse probing.
Conventional methods of detecting polymerase chain reaction (PCR) products require equipment and expertise which may not be available in diagnostic bacteriology laboratories, especially in developing countries. To this end we have examined other methods of product detection, including fluorescein-12-dUTP incorporation during PCR amplification, and reverse probing, where the PCR product is used as the probe in a scaled down hybridization with a fixed capture probe consisting of a fragment entirely internal to the sequence of the PCR product. These techniques have shown sensitivities of 20 fg of purified mycobacterial DNA, which corresponds to approximately five cells.
Woolford
A J
AJ
Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Dale
J W
JW
D001483:Base Sequence; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D003856:Deoxyuracil Nucleotides; D005069:Evaluation Studies as Topic; D005452:Fluoresceins; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004269:DNA, Bacterial; D003856:Deoxyuracil Nucleotides; D005452:Fluoresceins; C069632:fluorescein-11-deoxyuridine triphosphate