A rapid and simple method for labeling short DNA fragments using Taq polymerase.
This report describes a new method for labeling PCR-generated short length (60-120 bp) double-stranded DNA fragments for use as hybridization probes. The method utilizes gene-specific primers identical to those for PCR generation of non-radioactive DNA fragments. Radioactive probes are synthesized by Taq DNA polymerase without using PCR. Single-stranded (sense or antisense) and double-stranded probes can be individually prepared by selection of the appropriate primers. The labeling reaction reached maximum incorporation within 30 min with mean specific activities of 1.05 x 10(9) dpm/microgram (antisense single-stranded), and 1.62 x 10(9) dpm/microgram (double-stranded) were obtained using templates 69-117 of nucleotides. This method offers a simple and rapid means of generating antisense probes for Northern blot analyses and double-stranded probes for Southern blot analyses that provide highly intense signals with low background.
Mizobuchi
M
M
Dept. of Internal Medicine, University of Cincinnati, College of Medicine, OH 45267-0547.
Frohman
L A
LA
D000818:Animals; D001709:Biotechnology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D016373:DNA, Antisense / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005069:Evaluation Studies as Topic; D005260:Female; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D019914:Taq Polymerase
D015342:DNA Probes; D016373:DNA, Antisense; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase