Clontech PT3139-1 Benutzerhandbuch - Seite 6

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CLONTECH Laboratories, Inc.

I. Introduction continued

Automatic hot start with TaqStart Antibodies

Advantage-HF provides hot start PCR by including TaqStart

polymerase mix, eliminating the need for additional pipetting or handling steps.

"Hot start" refers to any method for assembling PCR reactions that keeps one or

more of the reaction components physically or functionally separate from the rest

of the components prior to the onset of thermal cycling. This prevents back-

ground due to low-level DNA synthesis from nonspecifically primed sites prior to

the onset of thermal cycling. The advantages of hot start PCR have been

demonstrated in many different applications. However, only CLONTECH's

Advantage Polymerase Mixes provide automatic hot start.

TaqStart is a neutralizing monoclonal antibody directed against Taq DNA

polymerase. TaqStart recognizes both native Taq and N-terminal deletions such

as KlenTaq-1. When premixed with the appropriate polymerase, the antibody

blocks polymerase activity during the set-up of the PCR reactions at ambient

temperatures. Polymerase activity is restored at the onset of thermal cycling

because the antibody is denatured by temperatures greater than 60°C. The loss

of inhibition is complete and irreversible, so the polymerase regains its full

enzymatic activity for PCR.

TaqStart-mediated hot start PCR has been shown to significantly improve the

efficiency and specificity of DNA amplifications (Kellogg et al ., 1994;

CLONTECHniques, April 1994). Antibody-mediated hot start with TaqStart has

been proven to be at least as effective as manual hot start (d'Aquila et al ., 1991)

or wax-bead-mediated hot start (Chou et al ., 1991). In particular, TaqStart

reduces or eliminates nonspecific amplification products and primer-dimer

artifacts. In some cases, specific products can only be obtained by using

TaqStart.

A PCR system for every application

The Advantage cDNA and Genomic PCR Kits, containing the Advantage cDNA

and Genomic Polymerase Mixes, respectively, are designed for high-efficiency,

long-distance PCR amplification and are the foundation of the Advantage PCR

Enzyme Systems ( CLONTECHniques , July 1995; Barnes, 1994). These versatile

kits are designed for high-performance amplification in the vast majority of PCR

applications (including all of our PCR-based kits) and have firmly established the

benefits of PCR enzyme mixes containing hot start antibodies. Our other Advan-

tage-based kits have been developed for certain specialized applications. The

Advantage-GC cDNA and Genomic PCR Kits and Mixes ( CLONTECHniques ,

January 1997) combine the high efficiency of the Advantage system with a novel

reagent, GC-Melt

cation of problematic GC-rich templates (up to 90% GC). As the newest addition

to the Advantage line, Advantage-HF allows a new level of fidelity, balanced by

the efficiency that is characteristic of Advantage.

page

Protocol # PT3139-1

Version # PR76834

, and an optimized buffer containing DMSO to permit amplifi-

Technical Support TEL:415-424-8222 or 800-662-CLON

FAX: 415-424-1064 or 800-424-1350

Antibody in the