ANALYTICAL BIOCHEMISTRY 255, 133–141 (1998) ARTICLE NO. AB972438 Thermal Cycle Labeling: Zeptomole Detection Sensitivity and Microgram Probe Amplification Using C viJI* Restriction-Generated Oligonucleotides Neela Swaminathan, 1 Karolyn McMaster, Piotr M. Skowron, and David A. Mead* CHIMERx, Madison, Wisconsin 53704; and *Molecular Biology Resources, Milwaukee, Wisconsin 53210 Received July 31, 1997 and variations of the polymerase chain reaction (PCR) A new method for efficiently labeling and amplifying (10). Nick translation replaces the nonlabeled nucleo- DNA probes from anonymous samples has been devel- tides of a polymer chain with labeled analogs by balanc- oped. The two/three base recognition endonuclease ing the activities of two enzymes, bovine DNase I and CviJI* restricts DNA to numerous small fragments pri- Escherichia coli DNA polymerase I. RPL utilizes oligo- marily 20–60 bp in size. Thermal denaturation of these nucleotides six to nine bases long (synthesized in all fragments results in sequence-specific oligonucleotides possible combinations) as primers for enzymatic incor- complementary to their cognate template. Repeated cy- poration of nucleotide analogs using a suitable DNA cles of denaturation, annealing, and extension of such polymerase, either the Klenow fragment of DNA poly- a multiprimed template by a thermostable DNA poly- merase I or T7 DNA polymerase. PCR methods for la- merase results in a significant amplification of the start- beling DNA require two opposing synthetic primers ing material. This method of amplification, referred to 20–30 bases long, labeled nucleotide analogs and a as thermal cycle labeling (TCL), appears to generate a thermostable DNA polymerase, such as the enzyme iso- large fraction of rearranged and presumably branched lated from Thermus aquaticus, in repeated cycles of products. The inclusion of nucleotide analogs in the denaturation, primer annealing, and extension (10). TCL reaction generates microgram amounts of hapten- The efficiency of these labeling methods depends on tagged probe with a detection limit of 25 zmol (2.5 1 reaction variables which can limit their detection sensi- 10 020 mol). Reactions containing [a- 33 P]dCTP yield high- tivities and hence their usefulness. For example, the specific-activity probes (2.6 1 10 9 cpm/mg) with reduced efficiency of labeling via nick translation is sensitive to radiolytic decay and a useful shelf life of 1 month. the ratio and age of the DNase I and DNA polymerase I CviJI*-generated primers circumvent the need for syn- (11). RPL requires microgram amounts of short random thetic oligos while providing microgram amounts of primers generally six to nine bases long. This length amplified and labeled probes using the described TCL of primer restricts the annealing temperature and protocol.  1998 Academic Press hence the enzymatic extension to less than 37°C, lim- iting this system to DNA polymerases such as Klenow or T7. The large amount of oligonucleotides required Labeled DNA probes are essential tools in numerous for RPL results in nonspecific labeling of the random hybridization-based applications such as colony and primers, as demonstrated by excluding the template plaque screening (1, 2), Southern (3) and Northern (4) from such a reaction, particularly with limiting blot hybridization, in situ hybridization (5), and se- amounts of template. Random primers longer than quencing by hybridization (6). Commonly used meth- seven bases have been shown to produce poor-quality ods to label DNA for such purposes are nick translation probes, due to extensive primer – primer annealing and (7), the random primer labeling (RPL) 2 method (8, 9), extension (12). PCR labeling methods can efficiently utilize small amounts of template but require some sequence information to chemically synthesize the ex- 1 To whom correspondence should be addressed at CHIMERx, 3802 Packers Avenue, Madison, WI 53704. Fax: (608) 244-6318. tension primers. In addition, the high concentration of 2 Abbreviations used: RPL, random primer labeling; NBT, nitro complimentary dsDNA probes generated from a PCR blue tetrazolium; DTT, dithiothreitol; TCL, thermal cycle labeling; labeling reaction can compete with the hybridization TBS, Tris-buffered saline; TCA, trichloroacetic acid; BSA, bovine se- rum albumin; BCIP, 5-Bromo-4-chloro-3-indolyl phosphate. to nucleic acid target. 133 0003-2697/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.