Methods and reagents: Fidelity of DNA polymerases for PCR
by Paul N. Hengen, Ph.D. *
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet. This month's column discusses the fidelity of polymerases used for
amplification of DNA by the polymerase chain reaction. For details on how to
partake in the newsgroup, see the accompanying box.
Discussions concerning the use of the polymerase chain reaction (PCR) for
cloning, and the fidelity of enzymes used for chemical amplification of DNA,
have occurred several times over the past year. Since the reviewers of many
journals don't seem to set any guidelines on how a sequence should be obtained,
publication of a research article may not necessarily hinge on whether the
determined sequence has actually been confirmed. Many new articles show
sequences derived only by PCR, and authors rarely state the number of attempts
made to determine the sequence.
Recently, a number of questions were raised on methds-reagents regarding how
much data should be used for determining what would be considered to be the
true sequence, and when it is clean enough to be entered into the repository of
known DNA sequences. Netters could not always agree on the best way to gain
sequence data or where exactly to draw the line for what should be acceptable
within the scientific community. Some people are more careful and have a
difficult time being convinced that a sequence is correct until several
sequencing attempts have been made. The derived sequence is then made from many
different clones and in both directions for confirmation. Others merely take
the first DNA sequence off the gel reader and consider it a done deal.
Although no exact rules exist, there seems to be an unspoken agreement among
scientists that the number of sequencing attempts should depend on the
circumstances of the individual experiments. In general, if the clone was
obtained by a single PCR, it is felt that sequencing three separate clones
would provide sufficient data for the correct sequence. However, if the
experiment involved a subset of the amplified material, as is the case for PCR
cloning, a single PCR experiment could give misleading information. It would
therefore be wiser to make several clone pools from different PCR attempts and
to sequence a number of these from each pool.
Some netters warn that more sequencing attempts should always be made when the
sequence has been derived from a single species within the pool of
PCR-generated DNA fragments, or when multiple steps are involved for the in
vitro extension of templates. For example, a reverse transcriptase PCR cloning
experiment should always have more confirming data than a sequencing project
involving a genomic library not generated by PCR.
In the course of cloning and sequencing by using PCR, the fidelity of the
polymerase used should be considered carefully. If an error occurs early on in
the PCR, that mistake could be propagated more than if it occurs during later
cycles, and it could find its way into a significant proportion of the clones
obtained.
Hi-fi PCR
The main problem with PCR is the fidelity, or lack thereof, of the various
polymerases under different conditions. Errors made by DNA polymerase can
affect the extension reaction of PCR during five distinct steps: (1) the
binding of the correct dNTP by polymerase; (2) the rate of phosphodiester bond
formation; (3) the rate of pyrophosphate release; (4) the continuation of
extension after a misincorporation; and (5) the ability of the enzyme to adjust
to a misincorporated base by providing 3'-to-5' exonuclease `proofreading'
activity. [1]
Misincorporation rates for different polymerases are described in terms of
errors per nucleotide polymerized, and the rate can be greatly affected by many
parameters. Several studies have concluded that different thermostable DNA
polymerases have error rates between 2.1 x 10^-4 to 1.6 x 10^-6 errors per
nucleotide per extension. [2-10]
However, error rates can vary for reasons other than those involving laxity of
the polymerase. For example, physical damage to DNA can result in
misincorporated bases, gaps and crossover products. To make matters worse, the
condition of the template is not the only concern; the source of the template
material can also affect fidelity - something not generally considered when
error rates of different polymerases are determined in different laboratories
and then compared.
Recently, Volker Knoop
(knoop_v@mpimg-berlin-dahlem.mpg.de)
wrote that template
DNA extracted from one species of hornwort greatly affected the ability of Taq
polymerase to amplify DNA by PCR. Even though DNA purified by
CsCl-ethidium-bromide was used and appeared quite clean by agarose gel
electrophoresis, lowered fidelity was apparently the cause of many
discrepancies when several clones were sequenced. It was not clear whether the
PCR failed because of a difficult stretch of DNA or because a high-GC region
stalled the polymerase, as has been shown in other cases. [11]
In an attempt to clarify the problem, samples of the bad template were added to
DNA-containing PCR mixtures that were from isolates extracted in the same way,
but from a different plant species. Since these samples were known to work
well, it became evident that the bad template contained a substance that
inhibited Taq polymerase. When PCR amplification finally succeeded after the
hornwort DNA had been diluted, sequencing of several clones suggested that
lowered fidelity was the cause of many descrepancies. It was therefore
concluded that some kind of inhibitor was present in the sample that affected
the fidelity of the polymerase when diluted.
Another netter had similar results with a different organism and could only get
consistent sequence data when an alternative purification method was used for
the extraction of template DNA. Since clean data were obtained routinely
during many other attempts using the same purification scheme and PCR
conditions, both netters feel that the loss of fidelity was a direct result of
contaminants co-purifying with their DNA samples, and that the error-prone
amplification is dependent on the species from which the DNA had come from.
Make your own Taq polymerase?
Can fidelity be improved by altering the purification method of DNA
polymerases? During this past month some netters were discussing the
hypothetical isolation and purification of Taq polymerase for in-house use.
This can be accomplished by following any one of the methods originally
described for the purification of Taq DNA polymerase I. (Refs 12-14). The
best purification scheme is probably that of Lawyer et al. [15], which outlines
the cloning, expression and purification of both the `Stoffel fragment' and
native forms of Taq DNA polymerase. This method is currently being used for the
large-scale commercial production of the enzyme.
Several strains of Thermus species can be obtained from the American Type
Culture Collection, as can the cloned gene for Taq DNA polymerase I (ATCC
40336) isolated from Thermus aquaticus YT-1 (ATCC 25104). These materials
are for research purposes only, however, and cannot be used for commercial
production of Taq.
Should you make your own polymerase? To do this would require a tremendous
effort and many netters think that the cost of doing it might not be worth all
the trouble, since high-quality enzyme is available from many sources for less
money and the price is now beginning to drop. Also, any improvements made in
fidelity might not be much better than that obtained with a commercial prep.
Improvements to the existing enzyme may be hindered because scientists are
sometimes uncertain about what is allowed and what is not according to patent
law. Many scientists are unclear of the legal problems they might encounter,
and some feel that they would not like to spend much of their time trying to
understand the legalese surrounding the patent of the PCR process. Research on
DNA polymerases to be used for PCR might infringe the current US patent, and
most scientists are unwilling to take on the legal problems of doing their own
polymerase purification while trying to avoid landing in a dangerous legal
battleground. [16]
Several netters warned that using home-grown Taq for PCR could be a poor
decision because the legal rights to PCR are owned by the Swiss company
Hoffmann-La Roche, Inc., and one person thought that because the production of
Taq polymerase is a patented process, the isolation of the enzyme would most
likely require a license from the patent holder. [16] By contrast, many people
who carry out PCR use alternative enzymes. Some netters feel very strongly
that, regardless of any legal claim to the PCR process, the isolation of Taq
polymerase from the naturally available T. aquaticus bacterium should not be a
violation of patent rights, but that the use of recombinant forms of the enzyme
that have been cloned and manipulated by biotechnology companies should be
considered an infringement of patent law.
Some netters feel that the scientific community as a whole owns the rights to
any such natural substance, and that any scientist should therefore be able to
produce enzymes for research purposes without paying royalties to any company.
They think that if they isolate enzymes for the purpose of doing PCR in their
own lab, rather than to be sold or used for commercial venture, they should not
be subject to license. [17]
Someone mentioned that any enzyme used for cycle sequencing, provided it is not
from T. aquaticus strain YT-1, is not included in the US patent, and could
therefore be used legally. Unfortunately, the use of thermal cycling for
amplification of DNA that is patented, and research using this technique is
covered by the US patent.
Good news for those wishing to grow their own Taq polymerase is that recent
developments in the fight over patent rights to PCR might change all this in
the future. For more information concerning the US patent on PCR, the PCR
patent applications (Patent Nos 05352600 and 4889818) can be downloaded from
the US patent database at http://town.hall.org/patent/patent.html. Additional
information concerning the dispute over patent rights to Taq polymerase can be
downloaded from http://www.promega.com.
References
[1] Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods Appl. 1, 17-24
[2] Barnes, W. M. (1992) Gene 112, 29-35
[3] Cariello, N. F., Swenberg, J. A. and Skopek, T. R. (1991) Nucleic Acids Res. 19, 4193-4198
[4] Cha, R. S. and Thilly, W. G. (1993) PCR Methods Appl. 3, S18-S29
[5] Eckert, K. A., and T. A. Kunkel (1990) Nucleic Acids Res. 18, 3739-3744
[6] Keohavong, P. and Thilly, W. G. (1989) Proc. Natl Acad. Sci. USA 86, 9253-9257
[7] Ling, L. L. et al. (1991) PCR Methods Appl. 1, 63-69
[8] Lundberg, K. S. et al. (1991) Gene 108, 1-6
[9] Mattila, P. et al (1991) Nucleic Acids Res. 19, 4967-4973
[10] Tindall, K. R. and Kunkel, T. A. (1988) Biochemistry 27, 6008-6013
[11] Cariello, N. F. et al. (1991) Gene 99, 105-108
[12] Chien, A., Edgar, D. B. and Trela, J. M. (1976) J. Bacteriol. 127, 1550-1557
[13] Kaledin, A. S., Slyusarenko, A. G. and Gorodetskii, S. I. (1980) Biokhimiya 45, 644-651
[14] Engelke, D. R. et al. (1990) Anal. Biochem. 191, 396-400
[15] Lawyer, F. C. et al. (1993) PCR Methods Appl. 2, 275-287
[16] Dickson, D. (1994) Nature 372, 212
[17] Sederoff, R. (1993) Science 259, 1521-1522
Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health.
Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author.
You found this at the World Wide Web (WWW) Uniform Resource Locator (URL):
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/aug95.txt
Any reference to this column must be cited as the following published
article:
@article{Hengen1995Augtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Fidelity of {DNA} polymerases for {PCR}",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "8",
pages = "324-325",
month = "August",
year = "1995"}
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******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* |
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