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
REPLY
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
1/16 Pfu or 1/50 Deep Vent
(by volume; 1/160 or
1/500 by units)
Full-length wild-type Taq
Only one enzyme
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
(set Robo block to
60 sec 95 C melt
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
(longer at later cycles)
3-10 min extensions
Hot start or Taq Antibody
start (for genomics) (b)
Cold start, no Antibody
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 1.4ul 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 33nucleotide 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 33nucleotide 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