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Epicentre Forum 6 (2)
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Haiying Grunenwald, Epicentre Technologies
Successful PCR depends on a variety of factors including the quality
of the template, choice of enzyme, and primer design, as well as the
amplification conditions used. The ideal PCR system would be able to:
1) consistently amplify a wide variety of templates including difficult
(e.g., high GC content or secondary structure) and long sequences, 2)
amplify with high fidelity, and 3) achieve amplification with little
optimization. While enzymes for high fidelity PCR exist, as do methods
for difficult and long PCR, no one system exists that addresses all of
these factors in an easy-to-use format.
Here, we introduce FailSafe™ PCR. The FailSafe PCR System
ensures successful and consistent PCR results with both routine and challenging
templates, including long templates (up to approximately 20 kb in length)
and templates with high GC content (>80% GC). The FailSafe PCR System
consists of two components. The FailSafe PCR Enzyme Mix is a unique enzyme
blend containing a 3' to 5' proofreading enzyme for high fidelity. PCR
products generated by the FailSafe PCR Enzyme Mix are readily cloned
with high efficiency in TA or blunt-end vectors. The second component
of the FailSafe PCR System is a set of FailSafe PCR PreMixes. The FailSafe
PCR PreMixes contain buffer, dNTPs, and various amounts of MgCl2 and
FailSafe PCR Enhancer (with betaine).* The user simply
adds template, primers, and the FailSafe PCR Enzyme Mix to the FailSafe
PCR PreMixes and amplifies. This single-step protocol is used for all
templates, no tedious optimization is required. The FailSafe PCR Enhancer
included in the PreMixes increases PCR specificity, sensitivity, and
consistency.
In this article, we compare the fidelity of the FailSafe PCR System
to other commercially available PCR enzymes and enzyme blends. We also
demonstrate amplification of long and difficult templates and multiplex
PCR using the FailSafe PCR System.
Fidelity of the FailSafe PCR Enzyme Mix
Applications of PCR such as cloning, expression, mutation analysis,
and long amplification require the use of enzymes with low error rates.
We compared the fidelity of the FailSafe PCR Enzyme Mix with enzymes
and enzyme blends from other suppliers using a PCR-based forward mutation
assay. The method is similar to that used by Cline et al.1 and
measures PCR fidelity by amplifying the alpha - complementing portion
of the lacZ gene and assessing its sequence integrity using a
blue/white colony screening assay. Amplification reactions were performed
using the reagents and protocols supplied by each respective manufacturer
and fidelity assays were performed side by side. As shown in Figure 1,
the fidelity of FailSafe PCR Enzyme Mix was at least three times better
than both standard Taq polymerases tested and was equivalent to or better
than the other high fidelity enzyme blends tested. Although the FailSafe
PCR Enzyme Mix exhibits slightly lower fidelity than has been reported
for Pfu DNA polymerase, the FailSafe PCR System is much more robust and
is able to achieve more specific and consistent amplification of difficult
templates (e.g., with high GC content) and long templates on which Pfu
fails.
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Figure 1. Fidelity comparison of FailSafe PCR Enzyme Mix with
other suppliers' enzymes or enzyme blends. |
Amplification of long sequences using the FailSafe PCR System
Amplification of long templates often requires tedious optimization
of reaction conditions including the addition of PCR additives. To demonstrate
that the FailSafe PCR System amplifies long templates, as well as standard
templates, without tedious optimization of individual reaction components,
we amplified lambda, human, and E. coli genomic DNA targets ranging
in size from 5 kb to 21.5 kb.
For each template/primer pair combination, PCR reactions were performed
using the FailSafe PCR PreMixes. Each 50 µl reaction contained
1-500 ng of genomic DNA (depending on the template), 10-50 pmoles of
each primer (depending on the template), 2.5 U of FailSafe PCR Enzyme
Mix, and 25 µl of a FailSafe PCR 2X PreMix (A-L). The lambda template
was amplified with the following cycling profile: 94°C for 1 minute,
followed by 20 cycles at 98°C for 20 seconds, 56°C for 1 minute
(5 kb and 10 kb only), and 68°C for 5 minutes (5 kb), 10 minutes
(10 kb), or 20 minutes (20 kb). The E. coli genomic DNA was amplified
with the following cycling profile: 94°C for 1 minute, followed by
20 cycles (except 30 cycles for the 6 kb template) of 98°C for 20
seconds and 68°C for 5 minutes (6 kb), 10 minutes (10 kb), or 20
minutes (18 kb). The human genomic DNA was amplified with the following
cycling profile: 94°C for 1 minute, followed by 14 cycles of 98°C
for 20 seconds and 68°C for 20 minutes, and then another 10 cycles
where the extension time is lengthened by 15 seconds for each subsequent
cycle.
Figure 2 shows the PCR products amplified using the optimal FailSafe
PCR PreMix for each template/primer pair combination. All PCR products
were amplified in high yield and with high specificity.
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Figure 2. Amplification of various sequence lengths using the
FailSafe PCR System. PCR amplification was performed as described
in the text. The results using the optimal FailSafe PCR PreMix
determined from each set of reactions is shown. M, 5 kb ladder. |
Amplification of GC-rich templates using the FailSafe PCR System
As with long PCR, amplification of difficult templates, such as those
with high GC content or secondary structure, often requires extensive
optimization. To demonstrate that the FailSafe PCR protocol can be used
for amplification of GC-rich templates without extensive optimization,
we amplified a 250-350 bp region of the human FMR1 gene, which has a
GC content of 80-85%.2 An expansion
of a triplet repeat (CGG) region in this gene is associated with fragile
X syndrome. Human genomic DNA was purified from blood with the MasterPure™ DNA
Purification Kit (Epicentre). PCR reactions were performed using the
FailSafe PCR PreMixes. Each 50 µl reaction contained 50 pmoles
of each primer, 100 ng of genomic DNA, 1.25 U of FailSafe PCR Enzyme
Mix, and 25 µl of a FailSafe PCR 2X PreMix (A-L). After an initial
denaturation at 94°C for 2 minutes, the Enzyme Mix was added and
the reaction was amplified at 94°C for 4 minutes, followed by 30
cycles of 98°C for 30 seconds, 65°C for 1 minute, and 72°C
for 1 minute. Using a single set of 12 reactions, FailSafe PCR PreMix
J was determined to be optimal for amplification of the high GC region
of the FMR1 fragile X gene (Figure 3).
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Figure 3. FailSafe PCR amplification of an 80-85% GC-rich region
of the human FMR1 gene. PCR was performed as described in the
text. Lanes A-L show the amplification products resulting from
PCR using the 12 FailSafe PCR PreMixes. M, molecular weight marker.
Optimal amplification was obtained with FailSafe PCR PreMix J. |
Amplification of this region of the fragile X gene from four separate
individuals was performed with FailSafe PCR PreMix J and the FailSafe
PCR Enzyme Mix. The results are shown in Figure 4. As seen in the Figure,
the FailSafe PCR System consistently amplified this 80-85% GC-rich region.
Because the number of CGG repeats varies among different individuals,
the size of the resulting PCR product varies slightly, ranging between
250 bp and 350 bp.
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Figure 4. FailSafe PCR amplification of the FMR1 region from
four different individuals. PCR was performed using FailSafe
PCR PreMix J as described in the text. M, 100 bp ladder. |
Multiplex amplification using the FailSafe PCR System
The FailSafe PCR System was also tested for multiplex amplification.
Five exons from the cystic fibrosis transmembrane conductance regulator
(CFTR) gene3 were amplified using
the FailSafe PCR PreMixes. The 50 µl multiplex PCR reactions contained
25 pmoles of each primer,2 500 ng
of human genomic DNA, 2.5 U of FailSafe PCR Enzyme Mix, and 25 µl
of a FailSafe PCR 2X PreMix. After an initial denaturation at 94°C
for 2 minutes, the Enzyme Mix was added and the reaction was amplified
for 30 cycles at 94°C for 10 seconds, 53°C for 10 seconds, 74°C
for 10 seconds, followed by a final extension step at 74°C for 5
minutes. The sizes of the PCR products from the CFTR gene exons 4, 10,
11, 20, and 21 are 423, 340, 233, 289, and 396 bp respectively.2 Optimal
amplification was achieved using FailSafe PCR PreMix C (Figure 5). The
multiplex analysis resulted in the correct size PCR product for each
exon. The 5-band multiplex PCR from the CFTR gene was successfully obtained
with one set of reactions using the FailSafe PCR System.
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Figure 5. Multiplex PCR amplification of the CFTR gene. Multiplex
PCR was performed as described in the text. The results using FailSafe
PCR PreMix C are shown. M, molecular weight marker. |
FailSafe PCR ensures successful PCR results for a variety of applications
including amplification of templates at least 20 kb in length, amplification
of GC-rich templates, and multiplex PCR. The high fidelity of the FailSafe
PCR Enzyme Mix is important for making PCR products for cloning, expression,
and mutation analysis. The convenient kit format enables easy, single-step
amplification of any template without tedious optimization, and no change
in protocol is required for different templates. These advantages make
the FailSafe PCR System suitable for both routine and challenging PCR
amplifications.
- Cline, J. et al. (1996) Nucl. Acids Res. 24 (18),
3546.
- Fu, Y.H. et al. (1991) Cell 67 (6), 1047.
- Richards, B. et al. (1993) Human Molecular Genetics 2 (2),
159.
*Patents issued and pending.
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