JMD 2002, Vol. 4, No. 2
Copyright © 2002 American Society for Investigative Pathology & Association for Molecular Pathology
Competitive Amplification and Unspecific Amplification in Polymerase Chain Reaction with Confronting Two-Pair Primers
Nobuyuki Hamajima*,
Toshiko Saito*,
Keitaro Matsuo*
and
Kazuo Tajima*
From the Division of Epidemiology and Prevention,
*
Aichi Cancer Center Research Institute, Nagoya; and Nagoya University Graduate School of Medicine,
Nagoya, Japan
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Abstract
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Polymerase chain reaction with confronting two-pair primers
(PCR-CTPP) is an inexpensive, time-saving genotyping method
that is applicable for most single nucleotide polymorphisms. To
date, we have applied PCR-CTPP successfully for the genotyping
of more than 30 polymorphisms. This paper demonstrates the differences
in DNA amplification among different annealing temperatures of PCR-CTPP
with given melting temperatures for four primers. The NQO1 C609T
(Pro187Ser) polymorphism was used as an example. Two sets of
four primers were applied for PCR-CTPP; the first set with different
melting temperatures (Tms), and the second with similar Tms.
The comparisons with one-pair primer PCR (allele-specific PCR) revealed
that PCR-CTPP amplified DNA more specifically than allele-specific PCR.
The primers with different Tms caused competitive DNA amplification for
heterozygous genotype. Four primers with similar Tms amplified both
alleles unspecifically at a lower annealing temperature, while
the same DNA samples were correctly genotyped under an optimal
annealing temperature. These findings are unique for PCR-CTPP,
and important characteristics when the primers and annealing
temperatures in PCR-CTPP are designed. The knowledge of these
characteristics will extend the applicability of PCR-CTPP for
polymorphism genotyping.
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Introduction
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Polymerase chain reaction (PCR) is the most common technique used
for genotyping, which has promoted studies on the associations of
genotypes with disease risk and prognosis.1, 2, 3, 4
Several PCR
methods are available and a method suitable to the nature of each study
is adopted for genotyping. Type of polymorphism, accuracy of
genotyping, number of samples, and available PCR equipment are factors
to be taken into account when a PCR method is chosen for a given study.
Genotyping for variable number of tandem repeat (VNTR) polymorphisms
can be conducted simply by PCR followed by electrophoresis, because DNA
products with different sizes specific to each allele are amplified by
PCR. PCR-RFLP (restriction fragment length polymorphism) requires
another step for incubation with a restriction enzyme before
electrophoresis.5
Selected restriction enzyme digests DNA
products with a particular base sequence, which enables it to
distinguish the allele with the particular sequence from the alleles
without it. It has been estimated that half of single nucleotide polymorphisms (SNPs) occur
in natural restriction sites.6
PCR-SSCP (single-strand
conformation polymorphism) is a genotyping technique to detect
differences in the conformation of single strand DNA,7
which requires electrophoresis equipment to keep a constant
temperature. Allele-specific PCR is an alternative for
genotyping,8
whose PCR condition is set to avoid
unspecific DNA products. The PCR method is conducted for each allele,
that is, for different alleles in different tubes.
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP)
is a new genotyping method invented independently,9, 10
which we recently found to be based on the same logic as bi-directional
PCR amplification of specific alleles (Bi-PASA) whose real applications
for genotyping have been infrequently reported.11
Both
methods for single nucleotide polymorphisms produce allele-specific DNA
bands with different lengths by adding four designed primers into one
tube containing an ordinarily prepared PCR mixture. The amplification
of allele-specific bands with different lengths makes genotyping
possible by electrophoresis without other steps. As shown in Figure 1
, the four primers consist of F1 and R1 for X allele amplifying a
a-bp DNA and F2 and R2 for Y allele producing a
b-bp DNA. F1 and R2 produce a c-bp common band.
R1 and F2 confront each other at the 3' end with the base specific to
the allele. The difference between the two methods is only in the
sequence design for the inner primers R1 and F2. Bi-PASA uses a
G+C-rich tail at the 5' end of the inner primers,11, 12
while our method does not.
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Figure 1. Logic of polymerase chain reaction with confronting two-pair primers.
At the 3' end of inner primers R1 and F2, the base specific to each
allele is designed. The difference between a-bp and
b-bp should be large enough to be distinguishable by
electrophoresis.
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There is no doubt that, if applicable, PCR-CTPP is an inexpensive,
time-saving method compared with PCR-RFLP and PCR-SSCP, although the
research groups applying this method for genotyping are still limited.
We have succeeded in the genotyping of more than 30
polymorphisms.13
This paper demonstrates the effects of
primer melting temperature (Tm) and PCR annealing temperature on the
DNA amplification. The effects are exemplified by NAD(P)H:quinone
oxidoreductase gene (NQO1)
C609T(Pro187Ser) polymorphism. The enzyme detoxifies
quinones and reduces oxidative stress. The T/T(Ser/Ser)
genotype causes complete loss of enzyme activity, which was reportedly
associated with acute myeloid leukemia, leukemia/myelodysplastic
syndrome,14, 15, 16, 17
colorectal cancer,18
and lung
cancer for smokers.19
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Materials and Methods
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Genotyping
DNA was extracted from 200 µl of buffy coat preserved at
-40°C by QIAamp DNA Blood mini kit (Qiagen Inc., Valencia, CA).
PCR-CTPP for the NQO1 polymorphism was conducted using two
sets of four primers. The first set of primers were as follows; F1:
5'-TAT CAG AGT GTC TTA CTG AGA (Tm at primer concentration 50 nmol/L
and salt concentration 50 mmol/L, 46.4°C) and R1: 5'- AAT GCT ATA TGT
CAG TTG AGG (51.6°C) for C allele amplifying a
161-bp band, F2: 5'-GTG GCT TCC AAG TCT TAG AAT (54.9°C)
and R2: TTT CTA GCT TTG ATC TGG TTG (54.5°C) for T allele
amplifying a 283-bp band. A 403-bp band was designed to be amplified
between primers F1 and R2. Although all of the primers were 21 mers
with 8 GC bases, the Tms estimated by a Tm prediction program
20
were different, as shown in the above parentheses. The
Tms for F2 and R2 were higher than those for F1 and R1. Four primers of
the second set had similar melting temperatures; F1': 5'-CCT TAT CAG
AGT GTC TTA CTG AGA (54.4°C) and R1': 5'-CAA TGC TAT ATG TCA GTT GAG
G (54.7°C), as well as F2 (54.9°C) and R2 (54.5°C).
F1' was three bases (CCT) longer than F1, and R1' one base (C) longer
than R1. Accordingly, the DNA amplified by the second set was 165 bp
between F1' and R1' and 406 bp between F1' and R2.
Genomic DNA (30 ng to 100 ng) was used in a volume of 25 µl with 0.18
mmol/L dNTPs, 12.5 pmol of each primer, 0.5 units of AmpliTaq Gold
(Perkin-Elmer Corp., Foster City, CA), and 2.5 µl of 10X PCR buffer
including 15 mmol/L MgCl2. GeneAmp PCR System
9700 (PE Biosystems, Foster City, CA) was used for PCR. The
amplification was conducted by 10 minutes of initial denaturation at
95°C, followed by 35 cycles of 1 minute at 95°C, 1 minute at
X°C, and 1 minute at 72°C, and a 5 minute final
extension at 72°C, where annealing temperature, X, was a
variable for this study. All PCR products were visualized on a 2%
agarose gel containing a 2 µl/100 ml of ethidium bromide.
The results of PCR-CTPP genotyping were confirmed by PCR-RFLP with
HinF1 enzyme, which produces 188-bp and 85-bp bands for
C allele and 151-bp and 85-bp bands for T allele
using primers F: 5'-AGT GGC ATT CTG CAT TTC TGT G, and R: 5'-GAT GGA
CTT GCC CAA GTG ATG.16
Forty-seven samples (18 for
C/C, 20 for C/T, and 9 for T/T) were
successfully genotyped, providing the same results as PCR-CTPP.
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Results
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Figure 2
shows the gels for the first set of primers (F1, R1, F2,
and R2) according to annealing temperature. DNA from three individuals
with a different genotype of NQO1 C609T was used for this
demonstration. At annealing temperature 59°C, one-pair PCR
(allele-specific PCR) with F1&R1 or F2&R2 amplified DNA for all of the
genotypes unspecifically, as shown in Figure 2
, lanes 1 to 3 and
7 to 9, respectively. PCR-CTPP produced a specific band for the
homozygous genotypes; a 161-bp band for C/C genotype (lane
4) and a 283-bp band for T/T genotype (lane 6). For
C/T genotype, two bands were amplified, but the strength of
the amplified bands was competitive, depending on annealing
temperature. At 59°C (Figure 2a)
the 161-bp band for C
allele was stronger, while at 64°C (Figure 2d)
the 283-bp band for
T allele was stronger. Figure 2b
at 61°C and Figure 2c
at 62°C was between them. Both allowed correct typing for the
three genotypes, but not at the other temperatures. In allele-specific
PCR, the allele specificity was observed between 61°C and 64°C for
F1 and R1, but not for F2 and R2.
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Figure 2. Gel showing the genotypes for
NAD(P)H:quinone
oxidoreductase gene
(NQO1),
C609T(Pro187Ser)
polymorphism by primers with different melting temperatures. Lane
M contains a 100-bp DNA ladder, lanes 1 to 3
for C allele primers (F1 and
R1), lanes 4 to 6 for four
primers (F1, R1, F2, and
R2), and lanes 7 to 9 for
T allele primers (F2 and
R2). PCR was conducted for template DNA from
three individuals with C/C, C/T, or T/T genotype,
from left to right. The annealing temperatures
were 59°C
(a), 61°C
(b), 62°C
(c), and
64°C (d).
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For the second set of primers with similar Tms (F1', R1', F2, and R2),
results performed at annealing temperatures 58°C and 63°C are
shown. At 58°C
(Figure 3a)
, PCR-CTPP amplified both alleles (lanes 4 to 6), which was useless
for genotyping, but PCR-CTPP at 63°C (Figure 3b)
produced an
acceptable specificity for the genotyping (lanes 4 to 6). Competitive
DNA amplification for C/T genotype demonstrated by the first
set of primers was not observed for the second set. When the annealing
temperature was set at 66°C, no observable bands were amplified.
Correct genotyping was possible at an annealing temperature between
62°C and 63°C.
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Figure 3. Gel showing the genotypes for
NAD(P)H:quinone
oxidoreductase gene
(NQO1),
C609T(Pro187Ser)
polymorphism by primers with a similar melting temperatures. Lane
M contains a 100-bp DNA ladder, lanes 1 to 3
for C allele primers (F1' and
R1'), lanes 4 to 6 for
four primers (F1', R1', F2, and
R2), and lanes 7 to 9 for
T allele primers (F2 and
R2). PCR was conducted for template DNA from
three individuals with C/C, C/T, or T/T genotype,
from left to right. The annealing temperatures
were 58°C
(a) and 63°C
(b).
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Discussion
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This paper has reported technical points for PCR-CTPP, which is
used only by a few research teams at present. However, we have
successfully designed PCR-CTPP conditions for more than 30
polymorphisms. These genotyping conditions will be reported in our
case-control studies elsewhere. There is no doubt that this method is
applicable to most single nucleotide polymorphisms.
The accuracy of PCR-CTPP for other polymorphisms has been confirmed for
IL-1B,21
ALDH2,22
FUT2, XRCC1, DRD2, and
L-myc by PCR-RFLP, and for IL-1B, ALDH2, and
FUT2 by DNA direct sequence. The DNA with a length expected
from the sequence itself indicates that the PCR-CTPP amplified the part
of polymorphism under examination, unless the highly homologous genes
exist. The problem of highly homologous genes relates not only to
PCR-CTPP, but also to PCR-RFLP and PCR-SSCP.
Through the process of finding a suitable condition, we learned that
the annealing temperature is more important in PCR-CTPP than in
PCR-RFLP. While PCR-RFLP requires a DNA amount enough for a digestion
step, PCR-CTPP needs balanced amplification between the two
allele-specific DNA products. If the actual Tm differs between the
two-pair primers, DNA is amplified competitively as seen for the first
set of four primers. When the Tm is similar among the four primers, the
DNA products are balanced. In such cases, a low annealing temperature
causes the unspecific amplification, which may be interpreted as
erroneous heterozygous genotype for all samples. In either case for the
Tm, an appropriate annealing temperature needs to be found by changing
the estimated Tm. Since we have found that, in many instances,
different Tms did not provide an optimal annealing temperature, we
prefer similar Tms for the four primers. The range of annealing
temperatures producing correct genotyping might be wider for primers
with similar Tms than for those with different Tms.
Until recently we used a Tm value calculated by a method based solely
on the number of bases and GC percentage for PCR-CTPP23
to
design primers with similar Tms. At that time it usually took more than
10 pilot PCRs to find a PCR-CTPP condition for clear genotyping. When a
nearest-neighbor algorithm based on a base sequence20
was
used for the Tm estimation, we found that there was a substantial
difference in the Tm for some primers between the two methods. All of
the primer sets by which PCR-CTPP failed accurate genotyping had
different Tm values, about 10°C of difference, even with the same
number of bases and GC percentage. Roughly 50% of the designed
PCR-CTPP had been aborted before establishing the condition; currently,
75% of the designed primers produce an acceptable level of accurate
genotyping. Although the actual Tm may still differ from the estimate
by the algorithm,24
the estimated Tm based on base
sequence is applicable to the primer design for PCR-CTPP. Since the Tm
is important for this method, further basic studies will be desirable
to establish a more comprehensive automated PCR-CTPP genotyping system.
Multiplex PCR is one of the promising applications for PCR-CTPP. We
established two sets of duplex PCR-CTPP; independent duplex PCR-CTPP
for IL-1B C-31T and IL-1RN VNTR polymorphisms,
and related duplex PCR-CTPP for the FUT2
gene.10
There are sets of polymorphisms to be genotyped at
the same time for predicting disease susceptibility, eg, cytochrome
p450s and glutathione S-transferases.25
The primer design
taking the Tm into account may produce a stable condition for multiplex
PCR-CTPP, which will accelerate studies of genetic
epidemiology.3
In conclusion, the advantages and disadvantages of PCR-CTPP have become
clear as the application increases. The competitive or unspecific
amplification is quite an interesting finding unique to PCR-CTPP, which
is one of the problems to disturb correct genotyping by PCR-CTPP. The
problem is simply resolved by choosing four primers with similar Tms
and by changing the annealing temperature. This method should quickly
replace PCR-RFLP, because the digestion step can be skipped, resulting
in lower costs and shorter genotyping times. For the researchers who
wish to design the PCR-CTPP conditions, the findings reported in this
paper are useful to avoid erroneous genotyping.
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Acknowledgments
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We thank Ms. Naomi Takeuchi for technical assistance.
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Footnotes
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Address reprint requests to Nobuyuki Hamajima, Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, 1–1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. E-mail: nhamajim{at}aichi-cc.jp
Supported in part by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports, Culture, and
Technology of Japan.
Accepted for publication December 3, 2001.
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Abstract
Introduction
