Clontech PT3139-1 User Manual

Advantage®-HF PCR Kit
User Manual

(PT3139-1)

Catalog #K1909-1, -y

Storage conditions: –20

°C

FOR RESEARCH USE ONLY

(PR76834)

CLONTECH Laboratories, Inc.

page Protocol # PT3139-1 Technical Support TEL:[phone] or 800-662-CLON
2 Version # PR76834 FAX:[phone] or 800-424-1350

Table of Contents

I. Introduction 3

II. List of Components 7

III. Additional Materials Required 8

IV. Advantage-HF PCR Kit Protocol 9

A. General Considerations 9
B. Control PCR Reactions 11
C. Recommended Cycling Parameters 12
D. Amplification of Longer Fragments with the Advantage Buffer 13
E. Recommendations for Electrophoresis 13

V. Troubleshooting Guide 14

VI. References 18

VII. Related Products 19

Notice to Purchaser

A license under U.S. patents 4,683,202, 4,683,195, and 4,965,188 or their foreign counterparts, owned by Hoffmann­La Roche and F. Hoffmann-La Roche Ltd. (“Roche”), has an up-front fee component and a running-royalty
component. The purchase price of this product includes limited, non-transferable rights under the running-royalty
component to use only this amount of the product to practice the Polymerase Chain Reaction (“PCR”) and related
products described in said patents solely for the research and development activities of the purchaser when this
product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to
the up-front fee component must be obtained by the end-user in order to have a complete license. These rights under
the up-front fee component may be purchased from Perkin-Elmer or obtained by purchasing an authorized thermal
cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting
the results of purchaser’s activity for a fee or other commercial consideration, is hereby granted by implication or
estoppel. Further information on purchasing licenses to practice the PCR process may be obtained by contacting
the Director of Licensing at the Perkin-Elmer Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404 or at
Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501.

This product is sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc.,
and the Perkin-Elmer Corporation.

Advantage-HF cDNA Polymerase Mix is covered by U.S. Patent No. 5,436,149. Foreign patents pending.
TaqStart Antibodies are licensed under U.S. Patent No. 5,338,671 and corresponding patents in other countries.

CLONTECH Laboratories, Inc.

TEL:[phone] or 800-662-CLON Technical Support Protocol # PT3139-1 page
FAX:[phone] or 800-424-1350 Version # PR76834 3

I. Introduction

The Advantage®-HF (High-Fidelity) PCR Kit is a KlenTaq-based system de­signed to deliver

Pfu

-like fidelity in the amplification of cDNA or genomic

templates.

High fidelity

and

efficiency

While

Pfu

polymerase is known for its exceptional fidelity, it suffers from subop­timal efficiency that can be problematic in many PCR applications. In contrast, the
Advantage-HF PCR Kit offers

Pfu

-like fidelity combined with the efficiency
required to amplify DNA fragments of up to 2.5 kb. These benefits are the result
of reformulation of several components in CLONTECH’s Advantage PCR
Enzyme Systems. The Advantage-HF Polymerase Mix combines KlenTaq (a 5'­exonuclease-deficient variant of

Taq

polymerase) with a proofreading poly­merase and TaqStartTM Antibody to provide a superior level of specificity.
Advantage-HF thus combines the benefits of

Pfu

and the Advantage Enzyme

System to deliver a high-fidelity enzyme system.

The HF Advantage

The accuracy of Advantage-HF is compared to other enzymes and enzyme mixes
in Figure 1. Using a genetic assay that measures nucleotide misincorporation,
Advantage-HF rivals

Pfu

in fidelity. This fidelity assay is based on amplification of

an

E. coli

ribosomal protein gene (Mo

et al.

, 1991). Mutations in this gene often
confer streptomycin resistance on the host. Upon introduction of the amplified
DNA into

E. coli

, the ratio of total transformants to streptomycin resistant

Figure 1. Comparison of fidelity of Advantage-HF and other PCR systems. The fidelity of
Advantage-HF compares favorably with that of the

Pfu

enzyme and is significantly higher than that of

other enzyme systems. Accuracy assay is based on 25 cycles of amplification (see text).

0

100

200

300

400

500

19

67

385

435

71

Taq Advantage AdvantageHFPfu

Taq

+

Pwo

Enzyme

Accuracy

(total transformants/strep

r

transformants)

CLONTECH Laboratories, Inc.

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I. Introduction

continued

transformants provides a comparative measure of PCR fidelity. The fidelity of
Advantage-HF was confirmed by sequencing (Table I). The high level of fidelity
delivered by the Advantage-HF system increases confidence in sequence de­rived from PCR products and is beneficial in a variety of PCR applications,
including expression studies of amplified full-length cDNAs, generation of cDNA
libraries, RACE, and analysis of homologous genes amplified with degenerate
primers.

TABLE I. FIDELITY OF ADVANTAGE-HF BASED ON SEQUENCING DATA

Error rate

a

Enzyme (per 100,000 bp)

Taq

180

b

Advantage-HF 2.4

a

determined with individual clones after 25 PCR cycles

b

agrees with published data (Ling

et al.

, 1991; Cariello

et al.

, 1991)

High-fidelity amplification of cDNA and Genomic templates

Advantage-HF was used to amplify several cDNA templates of different lengths
(Figure 2). Although amplification of the longest template yielded a reduced
amount of product (Lane 6), this amplified product contains a higher percentage
of accurate copies—nearly 6-fold higher than Advantage, and 20-fold higher than

Taq

, according to Figure 1.

Figure 2. Advantage-HF amplification of cDNA fragments. Several fragments were amplified from
Human Placenta cDNA under standard (Lanes 1, 3 & 5) and high-fidelity PCR conditions (Advantage­HF; Lanes 2, 4 & 6). M: λ/

Hin

d III DNA size markers. Lanes 1 & 2: 0.5-kb fragment of glycerol 3­phosphate dehydrogenase gene. Lanes 3 & 4: 1.3-kb fragment of transferrin receptor gene. Lanes 5
& 6: 2.5-kb fragment of lactoferrin gene. Cycling parameters: 30 sec at 94oC; 30 x (30 sec at 94 oC,
5 min at 68 oC); 5 min at 68 oC.

M123456

kb

4.4-

2.0-

0.56-

CLONTECH Laboratories, Inc.

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The Advantage-HF Kit ensures
high-fidelity amplification of ge­nomic as well as cDNA tem­plates. In Figure 3, the Advan­tage HF Kit was used to amplify
a 2.1-kb fragment of the bovine
pancreas trypsin inhibitor gene
from different amounts of total
calf thymus DNA. The fragment
was efficiently amplified from as
little as 25 ng of genomic DNA
(Lane 4).

Increase elongation efficiency
for longer fragments

Two reaction buffers are included
in the Advantage-HF Kit: the HF
Buffer and the original cDNA
Buffer. Use of the HF buffer de­livers the highest possible fidel­ity, as represented in Figure 1.
Fragments of up to ~2.5 kb can
be amplified under these condi­tions. To amplify longer frag­ments, some of the increase in
fidelity can be sacrificed to im­prove elongation efficiency by
combining the HF and cDNA
buffers in varying proportions.

Figure 4, Panel A shows the
fidelity resulting from the use of
varying percentages of the HF
Buffer (see p. 3 for description of
assay). Panel B demonstrates
amplification of a 6.0-kb cDNA
fragment using the indicated con­centrations of HF Buffer. In this
example, the fragment is suc­cessfully amplified in 80% HF
Buffer, conditions allowing a ~3­fold increase in fidelity over the
cDNA buffer. Optimal conditions
for the amplification of other frag­ments should be determined in­dividually.

I. Introduction

continued

Figure 4. The effect of HF Buffer concentration on PCR

fidelity (A) and amplification (B) of a 6.0-kb cDNA
fragment from Human Placenta cDNA. Size markers
are λ/

Hin

d III DNA.

Figure 3. Advantage-HF amplification from genomic
DNA. The indicated amounts of calf thymus DNA were

used to amplify a 2.1-kb fragment of the bovine pan­creas trypsin inhibitor gene. M: λ/

Hin

d III DNA size
markers. Cycling parameters are the same as in Fig­ure 2.

M 1 2345

ng: 100 75 50 25 0

B

200

40

60

80

100

0

0.5

1.0

1.5

2.0

HF Buffer %:

Mutants (%)

A

2.0-

4.4-

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Automatic hot start with TaqStart Antibodies

Advantage-HF provides hot start PCR by including TaqStartTM Antibody in the
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 back­ground 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 Advan­tage-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-MeltTM, and an optimized buffer containing DMSO to permit amplifi-

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.

I. Introduction

continued