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Focus: PCR Amplification
Long PCR Amplification of p300 cDNA Using a Blend of Taq DNA Polymerase and
Proofreading Pfu DNA Polymerase
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| Amplification of a large p300 cDNA was
accomplished using a combination of Taq DNA Polymerase and the
proofreading Pfu DNA Polymerase (Cat.# M7741). The
resulting 7.5kb DNA fragment was resolved on an agarose gel. Show Me the Data! |
By Joost Martens
MCB1, Leiden University Medical Center, Wassenaarseweg 72, Leiden, The Netherlands
Introduction
Transformation of cells by human adenoviruses is accomplished by
alterations in cellular gene expression. Essential for the transformation is the
adenovirus-E1A protein. The E1A protein exerts its effect by binding to cellular proteins,
like the retinoblastoma protein and the p300 protein. The p300 protein is known to be a
transcriptional coactivator for various transcription factors (1). In our studies we focus
on clarifying the role of the binding of E1A to p300 in the transformation process. For
this study, E1A and p300 expression constructs were needed. We used PCR(a) amplification as the first step in making these expression
constructs, as described for the 7.5kb p300 cDNA in this report.
It is generally known that amplification of DNA fragments larger than
1kb with Taq DNA polymerase is problematic. Lack of proofreading capacity allows
misincorporations, which reduce the efficiency of amplification. Amplification with a
proofreading thermostable DNA polymerase removes misincorporated nucleotides (2). However,
the yield of amplification product typically drops with increasing DNA length.
The combination of Taq DNA polymerase and a low level of
thermostable DNA polymerase that exhibits a 3´-exonuclease activity (proofreading DNA
polymerase, e.g., Pfu DNA polymerase) has been shown to successfully amplify DNA
fragments up to 35kb (2). This PCR amplification of large DNA templates results in
specific amplification products in sufficient amounts for downstream applications. This
approach has been applied to the 7.5kb p300 cDNA using a blend of Taq/Pfu
DNA Polymerase.
Long PCR Amplification
A DNA polymerase mix was made by combining 5 units of Taq DNA
Polymerase(b) and 0.2
units of Pfu DNA Polymerase(b) (Cat.# M7741). The
amplification reaction consisted of 1X Pfu DNA Polymerase Reaction Buffer, 250µM
of each dNTP, 1µM each of sense and antisense oligonucleotide primers, 50ng of plasmid
containing p300 cDNA and 1µl of the DNA polymerase mix in a total reaction volume of
50µl.
The amplification profile consisted of an initial denaturation at 94°C for 6 minutes
followed by 25 cycles of denaturation (94°C for 1 minute), annealing (50°C for 1 minute)
and extension (72°C for 16 minutes, approximately 2 minutes for every kilobase pair
amplified). A final extension of 8 minutes at 72°C was performed.
After amplification 10µl of the reaction mixture was tested on a 1% agarose gel
containing ethidium bromide. Amplified fragments were visualized by UV light. Fragment
size was estimated by inclusion of lambda Hind III/EcoR I markers.
Results
Figure 1 shows the results of the amplification. A fragment of the
predicted size (7.5kb) could be seen; no other fragments were observed.
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Figure 1. p300 PCR product following amplification
with Taq and Pfu DNA Polymerases. Lane M, 3µg lambda Hind
III/EcoR I markers; lane 1, 10µl of PCR product from the Taq/Pfu
DNA Polymerase) blend with degraded p300 cDNA clone;
lane 2, 10µl of PCR product from the Taq/Pfu DNA Polymerase blend with intact p300 cDNA clone. |
Discussion
Previously, PCR amplification of total p300 cDNA with Taq DNA polymerase was
hampered by the large size of the cDNA. Generally low or no yield is obtained and
mutations are introduced. However, the ability to PCR amplify long fragments with high
specificity would speed and simplify the analysis of the complete p300 cDNA. A plasmid
containing p300 cDNA was used as a template in a PCR amplification. Total length of this
plasmid was 13kb. Using the protocol described in this report, a 7.5kb fragment could be
amplified from this plasmid; this is the expected length of the p300 cDNA. Amplification
of the 7.5kb fragment with no apparent nonspecific amplification products suggests that
the procedure was specific for the target DNA.
Here we show the usefulness of a blend of Taq DNA Polymerase and the
proofreading Pfu DNA Polymerase to obtain long DNA templates with high
specificity. In this study a blend of 5 units of Taq DNA Polymerase and 0.2 units
of Pfu DNA Polymerase resulted in optimal amplification results.
References
- Eckner, R. et al. (1994) Molecular cloning and functional analysis of the
adenovirus E1A-associated 300-KD protein (p300) reveals a protein with properties of a
transcriptional adaptor. Genes Dev. 8, 869.
- Cheng, S. et al. (1994) Effective amplification of long targets from cloned
inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91,
5695.
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applicable in certain countries. Promega does not encourage or support the unauthorized or
unlicensed use of the PCR process. Use of this product is recommended for persons that
either have a license to perform PCR or are not required to obtain a license.
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are covered by patents issued and applicable in certain countries. Because purchase of
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product may be required to obtain a patent license depending upon the particular
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