Download Demonstration of the ExpandTM PCR System`s Greater Fidelity and

T E C H N I C A L
T I P S
BRUNO FREY AND BERNHARD SUPPMANN
B o e h r i n g e r M a n n h e i m G m b H , D e p a r t m e n t o f M o l e c u l a r B i o l o g y,
Nonnenwald, D-82372 Penzberg, Germany
Demonstration of the Expand PCR System’s Greater Fidelity
and Higher Yields with a lac I-based PCR Fidelity Assay
TM
lac I
lac Iq
lac Iq
Cut with Cla I
Ligate DNA
Transform
bacterial cells
➱
Grow cells on
agar plate
➱
8
Perform PCR
➱
lac Iq
Count blue colonies
and white colonies
Calculate error rate
Figure 1 Schematic of the lacI-based
fidelity assay.
Dra II 3632
3500
500
3000
lac Z'
Bla
Materials and Methods
Construction of pUCIQ17
The truncated lacI gene of pUC19 was
substituted by a functional copy of lacIq. A
178 bp Pvu II-Afl III fragment was replaced
by a 1121 bp DNA fragment encoding lacIq
(Figure 2). The α-complementing E. coli
strain DH5α (2), once transformed with the
resulting plasmid pUCIQ17 (3632 bp),
produces white (LACI+) colonies on LB
plates containing ampicillin (100 µg/ml)
and X-Gal (0.004% w/v).
q
➱ ➱ ➱
Thermostable polymerases possessing a
3'→5' exonuclease, or “proofreading,”
activity are often used in polymerase chain
reactions because they offer higher fidelity
amplification than enzymes lacking this
activity. Unfortunately, these proofreading
enzymes also produce substantially lower
amplification yields than polymerases
without proofreading activity. On the other
hand, “non-proofreading” DNA polymerases (e.g., Taq DNA polymerase, Tth
DNA polymerase) lack the high fidelity
required for applications in which accurate
replication is placed at a premium.
Coupled with a recent breakthrough in
PCR enzyme technology (1), these facts
have led to the development of the
Boehringer Mannheim ExpandTM PCR
System, optimized mixtures of Taq DNA
polymerase and the proofreading enzyme
Pwo DNA polymerase.
We wanted to demonstrate that the
Expand PCR System offers a better combination of high yields and high fidelity
amplification than non-proofreading
enzymes (e.g., Taq DNA polymerase) or
proofreading enzymes. Therefore, we performed a PCR fidelity assay (Figure 1) based
on the amplification, circularization, and
transformation of the pUC19 derivative
pUCIQ17, which contains a functional lacIq
allele (1). PCR-derived mutations in lacI
result in a de-repression of the expression of
lacZα and subsequent formation of a functional β-galactosidase enzyme, which can
be easily detected on X-Gal indicator plates.
pUCIQ17
3632
lac Iq
1000
2500
ori
2000
Figure 2
1500
Plasmid map of pUCIQ17.
Template preparation
pUCIQ17 was linearized by digestion
with Dra II. A typical PCR reaction contained 5 or 10 ng of linearized, gel-purified
plasmid DNA.
PCR primers
Both PCR primers used have Cla I
cleavage sites at their 5' ends:
Oligonucleotide CLA33 (34-mer, 24
matches: 5'-AGCTTATCGATGGCACTTTTCGGGGAAATGTGCG-3'), primes “left.”
Oligonucleotide CLA55 (36-mer, 26
matches: 5'-AGCTTATCGATAAGCGATGCCGGGAGCAGACAAGC-3') primes
“right” to the Dra II site of pUCIQ17
(pUC19). The length of the resulting PCR
product is 3,493 bp.
Polymerase chain reactions
The polymerase chain reactions were
performed according to the conditions
detailed in the manufacturers’ instructions.
We used the following cycling conditions
for all DNA polymerase preparations
except the Expand PCR System:
■ Denaturation: 10 s at 94°C
■ Annealing: 30 s at 57°C
■ Extension: 4 min at 72°C
for 18 cycles.
The following cycling conditions were
employed for the Expand PCR System:
■ Denaturation: 10 s at 92°C
■ Annealing: 30 s at 57°C
■ Extension: 4 min at 68°C
for 18 cycles.
Purification and circularization of the
3.5 kb PCR products
After the PCR, the amplification products were PEG-precipitated (without previous phenol treatment) according to Barnes
(3). After a restriction digest with Cla I, the
DNA was purified by gel electrophoresis.
BIOCHEMICA
■
NO.2 [1995]
T E C H N I C A L
Ligation reactions were carried out with the
Boehringer Mannheim Rapid Ligation Kit;
DNA concentrations per ligation reaction
did not exceed 30 ng.
lacI-based assay
The resulting PCR-derived plasmids
were transformed in E. coli DH5α as
described by Hanahan (2), and plated on
LB Amp100 X-Gal plates. After incubation
overnight at 37 oC, blue and white colonies
were counted. The error rate (f) per bp was
calculated with a rearranged equation published by Keohavong and Thilly (4):
f = -lnF / d x b bp;
where F is the fraction of white
colonies:
F = white (LACI+)/total colony number;
d is the number of DNA duplications:
2d = output DNA/input DNA;
and b is the effective target size of the
(1080 bp) lacI gene, which is 349 bp
according to Provost et al. (5); there are 349
phenotypically identified (by color screening) single-base substitutions (nonsense and
mis-sense) at 179 codons (approximately
50% of the coding region) within the lacI
gene (5). Frameshift errors, which may
occur at every position in the 1080 bp open
reading frame of lacI, are not taken into
account because little information is available for the specific DNA polymerases used
in PCR systems (except for Taq DNA polymerase, which has a frameshift error rate of
about 1/10th of the base substitution error
rate determined in a one-pass assay [6,7]).
Religation control
Fifty nanograms of Dra II-linearized, gelpurified pUCIQ17 DNA was religated, and
an aliquot of the ligation reaction was transformed into DH5α. After incubation
overnight, only 1 of 3750 growing colonies
(0.027%) showed a blue (LACI–) phenotype
on LB Amp X-Gal plates; therefore, formation of concatameric ligation products (with
subsequent intramolecular recombination in
E. coli that eliminates an additional origin of
replication) seems to be a very rare event.
Restriction analysis of PCR-derived plasmids
isolated from blue colonies also confirmed
that the LACI– phenotype originates in PCRderived mutations of lacI, but not in deleterious recombination events after transformation of the ligated DNA in DH5α.
BIOCHEMICA
■
NO.2 [1995]
Results
Table 1 illustrates that Boehringer
Mannheim’s Expand PCR System mixtures
offer better combinations of high amplification yield and high fidelity PCR. They
feature lower error rates than the enzymes
lacking proofreading activity, such as the
Taq DNA polymerase preparations and
Tth DNA polymerase. In addition, the
Expand PCR System mixtures produce
substantially higher yields than all proofreading DNA polymerases, even from
smaller amounts of DNA template. All
other commercially available polymerase
mixtures tested thus far demonstrated error
rates in the range of Taq DNA polymerase,
except the variation has been between
2.9 x 10–5 and 2.1 x 10–5 (results not
shown; publication in preparation).
The lacI-based fidelity system described
in this article represents a rapid, convenient
method for evaluating error rates for DNA
polymerases in PCR systems. Due to low
background activity, it is possible to measure error rates in the range of 10–4 to 10–6.
The calculated error rate for Taq DNA
polymerase is in good agreement with the
published value (8). ■
polymerase
template
amount
[ng]
5
yield [ng]
DNA
duplications (d)
9.2
Product
ExpandTM Long Template
PCR System*
(PCR 0.5–40 kb)
ExpandTM High Fidelity
PCR System*.†
(PCR ≤5 kb)
T I P S
Cat. No.
1681 834
Size
100 U
(30 reactions)
1681 842
500 U
(150 reactions)
1732 641
100 U
(30 reactions)
1732 650
500 U
(150 reactions)
See page 17 for local pricing.
†
Available after May 15, 1995.
References
1. Barnes, W. M. (1994) PNAS USA 91:2216.
2. Hanahan, D. (1983) J. Mol. Biol. 166:557.
3. Barnes, W. M. (1992) Gene 112:29.
4. Keohavong, P. and Thilly, W. G. (1989) PNAS USA
86:9253.
5. Provost, G. S., Kretz, P. L., Hamner, R. T., Matthews, C.
D., Rogers, B. J., Lundberg, K. S., Dycaico, M. J. and
Short, J. M. (1993) Mut. Research 288:133.
6. Tindall, K. R. and Kunkel, T. A. (1988) Biochemistry
27:6008.
7. Eckert, K. A. and Kunkel, T. A. (1990) Nucleic Acids
Research 18:3739.
8. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W.,
Short, J. M., Sorge, J. A. and Marthur, E. J. (1991)
Gene 108:1.
*Purchase of this product is accompanied by a limited license to use it in
the Polymerase Chain Reaction (PCR) process for life science research in
conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by
payment to Perkin-Elmer or as purchased, i.e., an authorized thermal
cycler.
blue
colonies
LACI–
151
white
colonies
LACI+
3066
total
% LACI–
number of
colonies
3217
4.7
error
rate
(ER565)
1.5x10–5
ExpandTM Long
3000
Template PCR
System1
ExpandTM High
5
3000
9.2
73
2695
2768
2.6
8.3x10–6
Fidelity PCR
System1
Taq from BM3
5
1500
8.2
167
2202
2369
7.1
2.6x10–5
Taq from
5
1500
8.2
148
1917
2065
7.2
2.6x10–5
Supplier C3
Tth from BM3
5
2000
8.6
280
2970
3250
8.6
3.0x10–5
Pwo from BM2
10
300
5
22
3447
3469
0.6
3.2x10–6
commonly used
10
80
3
12
2245
2257
0.5
3.8x10–6
proofreading
enzyme from
Supplier A2
(native enzyme)
commonly used
10
125
3.6
90
2527
2617
3.4
2.2x10–5
proofreading
enzyme from
Supplier B2
Table 1. Fidelity of different DNA polymerases in PCR. Enzyme preparations marked with a “1” are mixtures of
two polymerases, one of which possesses proofreading activity and one of which lacks proofreading activity.
Enzyme preparations marked with a “2” are single enzymes possessing proofreading activity. Enzyme preparations marked with a “3” are single enzymes lacking proofreading activity.
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