Download Reduced extension temperatures required for PCR amplification of

 1996 Oxford University Press
1574–1575 Nucleic Acids Research, 1996, Vol. 24, No. 8
Reduced extension temperatures required for PCR
amplification of extremely A+T-rich DNA
Xin-zhuan Su, Yimin Wu, C. David Sifri and Thomas E. Wellems*
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health,
Bethesda, MD 20892-0425, USA
Received February 14, 1996; Accepted March 4, 1996
A typical PCR cycle includes an extension step at 72C after
denaturation of double-stranded DNA and annealing of oligonucleotide primers. At this temperature the thermostable polymerase replicates the DNA at an optimal rate that depends on the
buffer and nature of the DNA template (1). Although the sizes of
the fragments that can be amplified have been generally limited
to <5 kb (2), recent reports have shown that a blend of two
polymerases (Taq + Pfu) allows replication and amplification of
much larger fragments, including a 42 kb sequence from the
bacteriophage λ genome (long PCR) (3,4). This ability to amplify
genomic DNA in vitro is of particular importance to studies of
Plasmodium falciparum, as large DNA fragments from this
malaria parasite are generally unstable in Escherichia coli (5). A
common finding with P.falciparum DNA, however, is that even
small fragments (<2 kb) can be difficult or impossible to amplify
under standard reaction conditions. Sequences that are refractory
to amplification often occur in the flanking regions of genes,
where the A+T- content can exceed 90% (6,7). Here we show that
reduction of the PCR extension temperature from 72 to 60C
allows amplification of this refractory A+T- rich DNA (>5 kb).
Figure 1a shows the effect of extension temperature on the PCR
products from four plasmid clones that contain A+T-rich
P.falciparum inserts of 1–2 kb (3F3, 6F9, 3E7, 7A6). PCR
amplification of each of the inserts was successful using an
extension temperature of 60, but not 65 or 72C. DNA sequences
were determined for three of the four inserts, and all were found
to have regions of ∼90% A+T-content extending for several
hundred bp. By comparison, the P.falciparum pfhsp86 coding
region, which supports PCR extension at 70C (8), has an
average A+T-content of 70% with only a single 100 bp region that
approaches 80%. We calculated the effect of these different A+T
contents on the melting temperatures (Tm) of the DNA sequences.
Figure 1b shows the predicted melting curves for representative
regions of the pfhsp86 coding region and the 3E7 insert. At a
cation concentration of 0.10 M and a DNA concentration of 0.1
pM, values that correspond approximately to conditions in the
early stages of PCR, the nucleotides of the pfhsp86 and 3E7
sequences have a 50% probability of being in an unpaired (open)
state at ∼73 and 64C respectively. The difference between these
temperatures corresponds to the empirically determined reduction in extension temperature necessary for the amplification of
the 3E7 sequence.
* To
whom correspondence should be addressed
Figure 1. Effect of temperature on the amplification and melting of A+T-rich
DNA sequences. (a) Results from DNA amplifications using PCR extension
temperatures of 60 and 65C. Reactions were performed in 50 µl volumes (0.5
ml tubes) containing 1 ng plasmid DNA, 25 pmol each M13 forward and
reverse primer (5′-GTAAAACGACGGCCAGT-3′, 5′-CAGGAAACAGCTATGAC-3′), 1 µl of 10 mM each dNTP, 5 µl 10× buffer (100 mM Tris–HCl/15
mM MgCl2/500 mM KCl pH 8.3, Boehringer Mannheim), and 1.5 U Taq
polymerase. After initial heating at 94C for 120 s, 20 cycles of PCR
amplification were performed, each consisting of four steps: denaturation at
94C for 20 s; annealing at 55C for 10 s followed by 50C for 10 s; and
extension at 60 or 65C for 120 s. Five microlitres of each amplification
reaction were loaded in the agarose gel (0.8%). (b) Temperatures at which
individual nucleotides of the 3E7 and pfhsp86 sequences are calculated to have
a 50% probability of the open (melted) state. Computations were performed
using 1000 bp sequences from the 3E7 insert (85% avg. A+T content) and from
part of the pfhsp86 coding region (70% avg. A+T content); results are shown
for bp 80–920 of each sequence. Temperatures were computed by the algorithm
of Poland (16) as implemented by Steger (17) (program POLAND available at
http://www.biophys.uni-duesseldorf.de/service/polandform.html). An ionic
strength of 0.10 M NaCl and a DNA concentration of 1.0 × 10–13 M were used
in the computations.
The success of reduced extension temperatures in the amplification of the 1–2 kb A+T-rich sequences suggested that these
temperatures are also important in the amplification of large (>5
kb) P.falciparum DNAs, as most DNAs of such size are expected
to have significant regions of >90% A+T content (including
intergenic regions and introns). We examined, therefore, the
effects of 60, 65 and 72C extension temperatures on the
amplification of different large P.falciparum DNAs. Figure 2
presents the results from one such series of experiments, a ‘long
PCR’ amplification of an 8 kb sequence from P.falciparum
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including those from other organisms as well as P.falciparum.
DNA replication at this reduced temperature appears to be
reliable and easily supported by the processivity of Taq polymerase. Indeed, routine use of 60C extension in our PCR protocols
has already produced a dramatic improvement in the successful
recovery of P.falciparum fragments, not only from standard and
long PCR amplifications, but from vectorette (9,10) and other
PCR methods (11–15) that are used to obtain regions flanking
known sequences. Reduced extension temperatures may also be
helpful in the application of cycle-sequencing methods to
extremely A+T-rich DNA.
ACKNOWLEDGEMENTS
Figure 2. Effect of extension temperature on the amplification of an 8 kb
P.falciparum DNA fragment. Reactions were performed in 50 µl volumes
containing 120 ng P.falciparum genomic DNA (Dd2) or water (H2O), 100 pmol
of each oligonucleotide primer (5′-GACTATTATTGTCACTATCC-3′; 5′-CCTAAAACCGACATCTTTTCC-3′), 5 µl of 10× Opti-Prime #6 buffer (100 mM
Tris–HCl/15 mM MgCl2/750 mM KCl pH 8.8), 1 µl of 10 mM each dNTP and
1.5 U TaqPlus polymerase (Stratagene). After initial heating at 94C for 120 s,
30 cycles of PCR amplification were performed, each consisting of four steps:
denaturation at 94C for 20 s; annealing at 52C for 10 s followed by 48C for
10 s; and extension at 72, 65 or 60C for 8 min. Five microlitres of each
amplification reaction were loaded in the agarose gel (0.8%). In the absence of
a specific band, high molecular weight smears of DNA were often found to
occur in these and other long PCR reactions, sometimes in the absence of added
DNA template (72C lanes).
chromosome 7. The bands show that the expected 8 kb fragment
was not obtained in PCR amplifications employing extension
temperatures of 72C, but it was obtained with an extension
temperature of 65C and, in even greater yield, with an extension
temperature of 60C. Amplification of a 7 kb fragment that
includes coding and flanking regions of the P.falciparum dhfr-ts
gene yielded similar results, i.e. product with PCR extension
temperatures at 60, but not at 65 or 72C (data not shown).
The results of these studies suggest that DNA melting prevents
Taq extension of extremely A+T-rich sequences at 72C. The
successful amplification of >5 kb fragments in this work further
suggests that a reduced extension temperature of 60C should be
routinely advised in the PCR of extremely A+T-rich sequences,
We thank David. S. Peterson and Kirk W. Deitsch for comments
on the manuscript.
REFERENCES
1 Innis,M.A. and Gelfand,D.H. (1990) In Innis,M.A., Gelfand,D.H.,
Sninsky,J.J. and White,T.J. (eds) PCR Protocols: A Guide to Methods and
Applications. Academic Press, Inc. San Diego, pp. 3–12.
2 Cheng,S., Chang,S.Y., Gravitt,P. and Respess,R. (1994) Nature, 369,
684–685.
3 Barnes,W.M. (1994) Proc. Natl Acad. Sci. USA, 91, 2216–2220.
4 Cheng,S., Fockler,C., Barnes,W.M. and Higuchi,R. (1994) Proc. Natl
Acad. Sci. USA, 91, 5695–5699.
5 Triglia,T., Wellems,T.E. and Kemp,D.J. (1992) Parasitol. Today, 8,
225–229.
6 Pollack,Y., Katzen,A.L., Spira,D.T. and Golenser,J. (1982) Nucleic Acids
Res. 10, 539–546.
7 Saul,A. and Battistutta,D. (1988) Mol. Biochem. Parasitol. 27, 35–42.
8 Su,X. and Wellems,T.E. (1994) Gene, 151, 225–230.
9 Arnold,C. and Hodgson,I.J. (1991) PCR Methods Appl. 1, 39–42.
10 Allen,M.J., Collick,A. and Jeffreys,A.J. (1994) PCR Methods Appl. 4,
71–75.
11 Triglia,T., Peterson,M.G. and Kemp,D.J. (1988) Nucleic Acids Res. 16,
8186.
12 Ochman,H., Gerber,A.S. and Hartl,D.L. (1988) Genetics, 120, 621–623.
13 Riley,J., Butler,R., Ogilvie,D., Finniear,R., Jenner,D., Powell,S., Anand,R.,
Smith,J.C. and Markham,A.F. (1990) Nucleic Acids Res. 18, 2887–2890.
14 Lagerstrom,M., Parik,J., Malmgren,H., Stewart,J., Pettersson,U. and
Landegren,U. (1991) PCR Methods Appl. 1, 111–119.
15 Jones,D.H. and Winistorfer,S.C. (1992) Nucleic Acids Res. 20, 595–600.
16 Poland,D. (1974) Biopolymers, 13, 1859–1871.
17 Steger,G. (1994) Nucleic Acids. Res. 22, 2760–2768.