Elsevier

Analytical Biochemistry

Volume 255, Issue 1, 1 January 1998, Pages 133-141
Analytical Biochemistry

Regular Article
Thermal Cycle Labeling: Zeptomole Detection Sensitivity and Microgram Probe Amplification UsingCviJI* Restriction-Generated Oligonucleotides

https://doi.org/10.1006/abio.1997.2438Get rights and content

Abstract

A new method for efficiently labeling and amplifying DNA probes from anonymous samples has been developed. The two/three base recognition endonucleaseCviJI* restricts DNA to numerous small fragments primarily 20–60 bp in size. Thermal denaturation of these fragments results in sequence-specific oligonucleotides complementary to their cognate template. Repeated cycles of denaturation, annealing, and extension of such a multiprimed template by a thermostable DNA polymerase results in a significant amplification of the starting material. This method of amplification, referred to as thermal cycle labeling (TCL), appears to generate a large fraction of rearranged and presumably branched products. The inclusion of nucleotide analogs in the TCL reaction generates microgram amounts of hapten-tagged probe with a detection limit of 25 zmol (2.5 × 10−20mol). Reactions containing [α-33P]dCTP yield high-specific-activity probes (2.6 × 109cpm/μg) with reduced radiolytic decay and a useful shelf life of 1 month.CviJI*-generated primers circumvent the need for synthetic oligos while providing microgram amounts of amplified and labeled probes using the described TCL protocol.

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