Life Science Quarterly January 2001

Jan 01 Jumpstart PCR

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Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 2   ISSUE 1   January 2001

 Molecular Biology Application Notes

JumpStartTM PCR -- Hot Start PCR Enzymes and Mixes

by Ernest Mueller, Erik Eastlund, and Keming Song
Sigma-Aldrich Corporation, St. Louis, MO, USA

Introduction

Polymerase Chain Reaction (PCR) Hot Start was first mentioned in the literature in 1991 by the inventor of PCR, Karey Mullis.1 Shortly thereafter, the practical advantages of Hot Start PCR were demonstrated in a 1992 publication by a group of Cetus application scientists.2 Hot Start is a PCR refinement that suppresses mis-priming artifacts and results in a more sensitive, consistent reaction with concomitantly higher yields. The technique works by either physically separating or chemically inactivating one or more of the reaction components until high temperature triggers mixing or reactivation to give a complete reaction mixture. Although the technique can be accomplished manually, the advent of high throughput PCR demands the convenience of a temperature-triggered delivery. The last ten years have seen a wide variety of Hot Start technologies become commercially available through a number of different vendors.

Discussion

Commercially available Hot Start methods employ either the wax barrier method3-5, a chemically modified Taq DNA polymerase6,7 or an antibody directed against the active site of the DNA polymerase.8 The most popular Hot Start technique uses Taq-directed antibodies since this method provides Hot Start compatible with high throughput PCR that is as effective yet more convenient than either the barrier techniques or chemically modified enzymes.8

The advantages of Hot Start can be readily seen when performing PCR with limiting template. Figure 1 shows the sensitivity gained by using a Hot Start PCR enzyme to amplify cDNA generated from the tobacco mosaic virus (TMV) genome. In most cases Hot Start increases sensitivity by 3-10 fold. Results are especially notable for reactions with template at 300 copies or below.8

Hot Start also gives better yields and greater specificity when amplifying difficult templates. This is especially true when amplifying bacterial or viral DNA to give amplicons greater than 5 kb, or genomic DNA with a target amplicon more than 3 kb, as shown in Figure 2.

The efficacy of antibody-mediated Hot Start is graphically depicted in Figure 3. This experiment uses a set of primers containing a 3' three base-pair homology to each other. After incubating for three hours at room temperature, and in the absence of exogenous template, the samples were subjected to PCR containing alpha32P]dCTP. As can be seen from the graph, increasing amounts of antibody suppress nucleotide incorporation. The accepted explanation for this result starts with the fact that unintended annealing (or mis-priming) is much more favorable at low temperatures. Once such annealing occurs, even though it may be transient, the primed double-strand is now a substrate for Taq DNA polymerase. Although low temperature conditions are not optimal for the enzyme, with enough time elongation of the mis-primed region will generate alternative primers. Subsequent PCR will amplify these "enhanced mis-primed" sites, diverting enzyme from production of the desired product. Thousands of side products will be generated in such reactions, which may not be produced in sufficient quantity to show on a gel but will manifest themselves by reducing the yield of the desired amplicon.

Our antibody-based Hot Start, marketed under the tradename JumpStartTM, increases the specificity and enhances yields without adjusting the cycling times often required by other methods. JumpStart PCR products are available in a variety of forms -- Taq and REDTaqTM DNA polymerase, ReadyMixesTM, and high-fidelity long and accurate blends.

Summary

Hot Start techniques can increase specificity (and yield) any time a complete reaction is incubated below the annealing temperature of the PCR primers. It can not, however, correct cycling reactions which have sub-optimal annealing steps, nor will it mediate the yield of products formed by oligo priming at homologous sites.

References and Note

1. Mullis, K.M., The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion. PCR Meth. Appl., 1, 1-4, (1991).
2. Chou, Q. et al., Prevention of pre-PCR mis-priming dimerization improved low-copy-number amplifications. NAR, 20(7), 1717-1723, (1992).
3. Bassam, B. J. and Caetano-Anolles, G., Automated "hot Start" PCR using mineral oil and paraffin. Biotechniques, 14(1), 31-34, (1993).
4. Riol, H. et al., A method of using heavy mineral oil for performing "hot-start" amplification of rare nucleic acids. Anal. Biochem., 221, 210-212, (1994).
5. Horton, R. M. et al., AmpliGrease: "hot start" PCR using petroleum jelly. BioTechniques, 16(1), 42-43, (1994).
6. Birch, D. E. et al., Simplified hot start PCR. Nature 381, 445-446, (1996).
7. Kebelmann-Betzing, C., et al., Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR. BioTechniques, 24(1), 154-158, (1998).
8. Kellog, D. E. et al., TaqStart Antibody': "Hot Start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques, 16(6), 1134-1138, (1994).

*Purchases of these products are accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose in the automated performance of the PCR process is covered by the upfront license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. These products are sold under licensing arrangements with F. Hoffmann-LaRoche Ltd., Roche Molecular Systems, Inc. and Perkin-Elmer Corporation.

About the Authors

Ernest Mueller, Ph.D., is a senior scientist and Erik Eastlund, M.S., is a scientist in PCR R&D at Sigma-Aldrich, St. Louis, MO. Keming Song, Ph.D., is a research fellow in Recombinant Protein Expression R&D at Sigma-Aldrich, St. Louis, MO.

ORDERING INFORMATION
Product Code Product Name Unit
D9307 JumpStart TaqTM DNA Polymerase 50 units
    250 units
    1,500 units
D4184 JumpStart TaqTM DNA Polymerase without MgCl2 50 units
    250 units
    1,500 units
D8187 JumpStart REDTaqTM DNA Polymerase 50 units
    250 units
    2,500 units
D1313 JumpStart REDAccuTaqTM LA DNA Polymerase 50 units
    250 units
    2,500 units
SUPPORTING LITERATURE
PCR Brochure (BKR)
Life Science Catalog 2000-2001, pages 30-35

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