Methods and reagents: Long and accurate 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 some problems encountered during the
amplification of large DNA segments by the polymerase chain reaction (PCR),
and gives tips for doing long and accurate PCR. For details on how to partake
in the newsgroup, see the accompanying box.
A recent discussion came about of a technique for amplifying very large
fragments of DNA with high fidelity, developed by Wayne M. Barnes
(Barnes@biolgy.wustl.edu)
at the Washington University School of Medicine, St. Louis [1].
Barnes proposes that the limiting factor in long-range PCR is premature
termination of the extension product owing to misincorporation of nucleotides.
The long and accurate (LA) PCR, or `Barnesian' method, employs a mixture of two
thermostable DNA polymerases, one that is highly processive and one with 3' to
5' exonuclease activity, allowing `proofreading' of the product.
After studying different combinations of enzymes, including Amplitaq/Pfu,
KlenTaq1/Vent, KlenTaq5/Pfu, Stoffel/Pfu, KlenTaq1/Deep Vent and Pfu exo-/Pfu
exo+, it was found that the best combination for obtaining PCR products as
large as 35 kb was a 16:1 ratio of KlenTaq1, an exonuclease-free mutant of Taq
polymerase, and Pfu polymerase, which has 3'-exonuclease actvity. The mixture
was termed KlenTaqLA-16.
Unfortunately, despite the more recent publication of conditions for reliable
`long PCR' [2], some people unaccustomed to Wayne's world of LA PCR are trying
to use enzyme combinations without much success.
Instead of distinct bands of expected PCR products, a smear is commonly seen
extending the length of the gel when a portion (25-50 ul of a 50 ul reaction
mixture) of the amplified material, either from complex genomic DNA or from
very large fragments cloned within cosmid vectors, is electrophoresed through
an agarose gel, stained with ethidium bromide and visualized under UV
illumination.
Smears were seen by netters regardless of the enzyme combination used. One
reported seeing the proverbial smear every time a combination of Taq and Vent
polymerases was tried, but that a faint band of DNA the correct size for the
expected amplified product was also visible behind the background of smeared
DNA. Another person could not see any bands when attempting to amplify a
fragment of 12 kb from a cosmid clone using the KlenTaqLA-16 combination,
even though this is much smaller than the largest product obtained by Barnes.
Interestingly, the smears appear even in control samples lacking DNA template,
suggesting that the material within the smear may be partially composed of
amplified contaminating DNA, complexes of primer and enzymes, primer-dimer
concatamers or coagulates of buffer components.
One person wrote that concatamers of primers alone are an unlikely source since
they would have to have grown extremely large during the course of the
experiment to account for the breadth of the smear within the gel. Others are
convinced that the culprit is bovine serum albumin (BSA), added by companies to
enyme stocks to protect the enzyme activity, or supplied in concentrated PCR
buffers. It was also mentioned that the use of gelatin in place of the BSA
might circumvent the problem.
One way of lessening the amount of smearing is to heat the reaction mixture to
95 degrees C for five minutes before adding the polymerase. This may destroy
the BSA or any other contaminating proteins, or may allow more specific
annealing of primers. On the other hand, it might cause more trouble, because
the extended denaturation step may damage the DNA and thwart amplification of
large strands. Some people have also tried heating their amplified products to
65 degrees C for five minutes before loading onto an agarose gel, in the hope
of disrupting the smear-causing complex.
Others suggested that a higher number of PCR cycles (25-30) with genomic DNA as
template may lead to incompletely extended DNA strands, causing the diversity
of DNA lengths that would appear as a smear. Lowering the number of cycles
should then relieve the smearing. One person reported that samples taken every
few cycles result first in the appearance of the expected DNA fragment,
followed by larger-sized bands, and finally smears. However, another netter
who removed samples every five cycles, noticed that the only difference seen
over the course of many cycles was a longer smear.
After all the tweaking of PCR conditions, netters keen on amplifying large DNA
fragments are frustrated from not being able to get clean results.
References
[1] Barnes, W. M. (1994) Proc. Natl. Acad. Sci. USA 91,2216-2220
[2] Cheng, S. et al. (1994) Proc. Natl. Acad. Sci. USA 91,5695-5699
Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA
e-mail: pnh@ncifcrf.gov
Tips and tricks for long and accurate PCR
Although I will admit to experiencing some problems recently (and describe a
promising cure; Table I c), I would first like to defend my method against
those who do not follow it, yet complain that it doesn't work.
The conditions for LA PCR are very narrow at this stage of development. I am
always bumping into the edges of the useful window of conditions, and I often
thank my lucky stars that I hit the window in my first few experiments, or else
I would have concluded that there was nothing there, and would not have found
the `long' conditions.
Anyone attempting to do research on this method should, at least for now,
follow every detail strictly. One person (an NIH virologist from Quebec) has
called me saying that he did this, and he is very happy, having immediately
reproduced my 35 kb amplicon. He says that his lab always uses filtered tips,
and has separate areas for reaction setup and gels, so he is all set to make
new progress. Netters who do not follow my recipe exactly and get poor results
are just repeating experiments that I have done and (mostly) reported.
If you wish to join me in doing research on the method, you should choose
conditions listed in the first column of Table I. This method may not yet be
ready for routine application, but fully-licensed formulae should be available
commercially within three months. If you are using something from the second
column, even any single item above the last three new ones, expect less
success. By less success I mean that amplifying 6-8 kb will work, but
amplifying 20 kb and above will result in decreased yields or worse.
Table I. The way to successful LA PCR
______________________________________________________________________________ LA PCR Classical PCR ______________________________________________________________________________ Klentaq1 (a) plus Full-length wild-type Taq 1/16 Pfu or 1/50 Deep Vent Only one enzyme (by volume; 1/160 or 1/500 by units) Thin-walled tubes Original-thickness tubes RoboCycler P.E. original or 480 cycler pH 9.2 pH 8.3 16 mM (NH4)2SO4, no KCl 50 mM KCl 50 mM Tris 20 mM Tris 3.5 mM MgCl2 1-2 mM MgCl2 5 sec 95 C melt 60 sec 95 C melt (set Robo block to 99 C for 30-40 sec) 2 ng lambda DNA 20 ng lambda DNA 33 ul reaction volume 100 ul reaction volume 20 cycles 30 cycles 68 C extension temperature 72 C extension temperature 33-nucleotide primers 20-22-nucleotide primers 11-24 min extension 3-10 min extensions (longer at later cycles) Hot start or Taq Antibody Cold start, no Antibody start (for genomics) (b) Filter tips (c) Non-filter tips UVA + 8MOP before template (d) No treatment ______________________________________________________________________________ (a) The level of Klentaq1 should be titred in 0.2 ul increments from 0.6 to 1.4 ul per 100 ul reaction, or at least from 0.8 to 1.2 ul. When I stated in my paper that Taq/Pfu shows the LA effect, I meant that Taq benefits from added Pfu, but, as stated, it does not, in my hands so far, come up to the performance of Klentaq1/Pfu. (b) The high pH, auto-extend cycles and hot start are from the adaptation by S. Cheng [1,2] at Roche. As single substitutions from her system into my system, I have not yet found the following to work above 20 kb: Taq, Tth, DMSO, glycerol, 9600 cycler, 22-nucleotide primers. Apparently, it is essential to go all the way in the direction of either system. I am not yet claiming any success with genomic samples above a few kilobases, but Suzanne Cheng can amplify up to 22 kb from human DNA. (c) The problem I have had (and cured, I believe) with my own method is that primers and targets, from 11 kb up to 35 kb, work fine only for a few weeks to months. Then they begin to fail more-or-less regularly, depending on the experience of the person doing the experiments. Since I have switched to filtered tips (which cost 8 cents each), and since I began to chlorox my pipettor barrel weekly, this problem has apparently gone away. I remade all of my stocks using the new tips, sometimes working at a sterile bench. The reason for the `bad' reactions may be carry-over of a `bad seed'. This bad seed is made in small quantities even in a good reaction, and if it gets into the air of a pipet barrel, thence into the dNTP and/or primer stocks, the bad seed recruits good product into the unidentified stuff, described in my paper, at the top of some of the agarose-gel wells. Originally, I had been using one pipet for everything, from making or dividing reactions before the PCR to loading the gel. The bad-seed reaction takes over above cycle 16, so it can usually also be avoided by doing no more than 16 cycles. I am now unsure of the proper conclusion for the 27-nucleotide versus 33-nucleotide experiment in my paper. Perhaps the proper conclusion is not `27-nucleotide work better than 33-nucleotide primers', but `New primers work better than old primers (that have been used for a while and whose bad seed is everywhere)'. I am still recommending 33-nucleotide primers until I know for sure. (d) Treatment of the enzyme(s) with 8-methoxy-psoralen (8MOPS) and UVA radiation [3] seems to be a good idea for inactivating endogenous contaminating bacterial DNA, but I am just trying it now.
References
[1] Cheng, S., Fockler, C., Barnes, W. M. and Higuchi, R. (1994) Proc. Natl. Acad. Sci. USA 91,5695-5699
[2] Sharkey, D. J. et al (1994) Bio/Technology 12,506-510
[3] Jinno, Y., Yoshiura, K., and Niikawa, N. (1990) Nucleic Acids Res. 18:6739.
Wayne M. Barnes
Department of Biochemistry 8231
Washington University Medical School
St. Louis, MO 63110, USA
email: Barnes@biolgy.wustl.edu
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/aug94.txt
Any reference to this column must be cited as the following published
article:
@article{Hengen1994Augtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Long and accurate {PCR}",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "8",
pages = "341-342",
month = "August",
year = "1994"}
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