Clinical Chemistry 43: 759-763, 1997;
(Clinical Chemistry. 1997;43:759-763.)
© 1997 American Association for Clinical Chemistry, Inc.
Analysis of a polyadenine tract of the transforming growth factor-ß type II receptor gene in colorectal cancers by non-gel-sieving capillary electrophoresis
Michiei Oto1,2,a,
Akira Koguchi3 and
Yasuhito Yuasa2
1
Department of Biotechnology, Tokyo Technical College, 1-15-5 Higashi, Kunitachi-shi, Tokyo 186, Japan.
2
Department of Hygiene and Oncology, Tokyo Medical and
Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo 113,
Japan.
3
Nippon Bio-Rad Laboratories, Higashi-Nippori,
Arakawa-ku, Tokyo 116, Japan.
a Author for correspondence. Fax 81-425-74-0184; e-mail wh6m-ootu{at}asahi-net.or.jp
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Abstract
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We developed a method to analyze a polyadenine tract, the
(A)10 repeat, within the cysteine-rich domain of the
transforming growth factor-ß (TGF-ß) type II receptor gene using a
non-gel-sieving capillary electrophoresis technique and applied it to
the DNA diagnosis of colorectal cancers. This method consists of
single-strand DNA amplification of the (A)10 repeat by an
asymmetric PCR technique and capillary electrophoresis. A higher
concentration of dATP in the PCR reaction mixture led to more specific
amplification of the (A)10 repeat. Under the optimal
electrophoretic conditions, one nucleotide difference could be
determined in 8 to 32 nucleotides. One or two base deletions of the
(A)10 repeat in colorectal cancers could be detected under
these conditions within 30 min, and the results coincided with those
obtained on DNA sequencing analyses. According to a sensitivity study,
we could detect the deleted sequence if it was present in 12.5% or
more of the wild-type allele. The reproducibility of this technique was
satisfactory because the intraassay imprecision (CV) (n = 10) was
1.4%. These results indicate that capillary electrophoretic analysis
of small repeated sequences results in easier handling and more
feasible automation, compared with conventional gel electrophoretic
analysis.
Key Words: indexing terms: polymerase chain reaction • DNA diagnosis
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Introduction
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Microsatellite instability is a DNA diagnostic landmark effective
not only for cancers, but also for various genetic diseases
(1)(2)(3)(4)(5)(6). In cancer development, this phenomenon is clearly
brought about by defects of a group of DNA mismatch repair genes,
hMSH2, hMLH1, hPMS1, hPMS2,
and GTBP [7]. However, the role of the microsatellite
instability in tumorigenesis remains unclear (8).
Transforming growth factor-ß (TGF-ß) is a multifunctional factor
that regulates cell differentiation and the expression of extracellular
matrix proteins, and also inhibits the growth of many cells, including
epithelial cells
(9).1
This growth-inhibitory signal is transduced through two
distantly related transmembrane serine/threonine kinases called
receptor types I and II (10). When these receptors are
inactivated, the cells lose their responsiveness to TGF-ß, and this
loss of growth inhibition by TGF-ß may be an important step in the
tumorigenesis of some cancers (11)(12). One or
two base deletions of a polyadenine tract, the (A)10
repeat, of the TGF-ß type II receptor (TGF-ß RII) gene
reportedly were detected recently in 90% of hereditary nonpolyposis
colorectal cancers and some sporadic colorectal cancers with
microsatellite instability (13)(14)(15), indicating that the
(A)10 repeat is a hot spot of mutation. Therefore,
TGF-ß RII may be one of the target genes of defective DNA
repair, and analysis of the (A)10 repeat is a good landmark
in the DNA diagnosis of cancers.
We combined PCR amplification and gel electrophoretic analysis to
analyze microsatellite DNA. However, for routine DNA diagnosis, the
development of an automated analytical system is indispensable.
Automated DNA extraction methods and robotic manipulation have been
developed for pre-PCR steps, and relevant materials are commercially
available. Generally, for post-PCR steps, gel electrophoresis followed
by ethidium bromide or silver staining separate and detect
nanogram–picogram quantities of DNA (16). However, gel
electrophoresis is not suitable for routine diagnosis, because it is
difficult to automate for many specimens.
In contrast to gel electrophoresis, capillary electrophoresis (CE)
reportedly is a promising technique for performing rapid and
reproducible separation of double-stranded DNA (dsDNA) as well as
single-stranded DNA (ssDNA), and its use is feasible for automated DNA
analysis (17). Non-gel-sieving CE is a specialized form of
capillary zone electrophoresis performed with a buffer containing
polymer additives such as methylcellulose (18). In
non-gel-sieving CE, the buffer containing polymer is packed
automatically into a capillary, and handling is easier than in the case
of gel-sieving CE (18). In recent reports, non-gel-sieving
CE has been applied to various DNA diagnostic techniques including both
hybridization and single-strand conformation polymorphism (SSCP)
analyses (19)(20). Here, we developed a method
for (A)10 repeat analysis of TGF-ß RII
involving non-gel-sieving CE and applied it to the DNA diagnosis of
colorectal cancers.
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Materials and Methods
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dna templates
Surgical specimens were obtained from colorectal cancer patients
according to the procedures of the Helsinki Declaration of 1975, as
revised in 1983. High-molecular-mass DNA from normal mucosa and tumor
tissue of the same patient was extracted by the method described
previously (21). Lymphocyte DNA from the respective
patients was also prepared similarly.
dna amplification with an asymmetric pcr technique
ssDNA including the (A)10 repetitive sequence of
TGF-ß RII was amplified by a modified asymmetric PCR
technique under optimal conditions with specific primers (Fig. 1
). Oligonucleotide primers were synthesized by the
phosphoramidite method with Cyclon Plus (Millipore, Bedford, MA).
Genomic DNA was subjected to asymmetric PCR.
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Figure 1. Primers used for the amplification of the
(A)10 repeat within the TGF-ß RII gene
(A); amplification of the (A)10 repeat by the
asymmetric PCR technique (B).
The primer sequences (20 nt) are indicated in open boxes
(A). Open boxes show the specific primers
(B). The dATP-rich nucleotides give a specific single-strand
DNA (30 nt) containing the (A)10 repeat.
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Asymmetric PCR was performed in a thin-walled tube (Robbins Labs.,
Sunnyvale, CA). The oligonucleotide forward primer (500 nmol/L),
reverse primer (50 nmol/L), dATP (200 mmol/L), dGTP (20 mmol/L), dCTP
(20 mmol/L), dTTP (20 mmol/L), and Taq DNA polymerase (0.5
unit) (Wako Chemical Co., Osaka, Japan) were mixed in a solution
composed of 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 1.5 mmol/L
MgCl2, and 1 g/L gelatin, and then a piece of wax
(AmpliWaxTM; Perkin-Elmer Instruments Co., Norwalk, CT) was
added. To melt the wax, the tube was kept at 70 °C for 1 min and
subsequently at room temperature to solidify the wax. To obtain
constant results and for application to routine assaying, the
preparations were stored in multiple tubes at 4 °C until the PCR
experiments. After 20–100 ng of the template DNA were placed on the
wax in the stored tubes, 40 cycles of 94 °C denaturation for 10
s, 55 °C annealing for 10 s, and 60 °C extension for 10
s were carried out in a program incubator (Zymoreactor II; ATTO Co.,
Tokyo, Japan).
analysis of pcr products (ssdna) by non-gel-sieving ce
The solid wax was pierced with a pipette tip, and the amplified
products were taken out, denatured at 94 °C for 5 min, chilled on
ice, and subjected to non-gel CE directly without further purification.
CE was performed with a BioFocus 3000TM CE apparatus
(Bio-Rad Laboratories, Hercules, CA). The capillary cartridge contained
a 50 µm (i.d.) x 36 cm polyacrylamide-coated capillary. Before
sample migration, the running buffer was filtered and the samples were
degassed by centrifugation. Pressurized injection at 414 kPa for 1
s was performed to introduce 10 nL of a DNA sample for analysis. DNA
samples were electrophoresed for 40 min at 500 V/cm at 25 °C in PCR
Product Analysis BufferTM (Bio-Rad Labs). The ultraviolet
detector was set at 260 nm, with a range of 0.02 absorbance units. PCR
product sizes were determined by comparison with standard nucleotides
(Pharmacia Biotech Co., Uppsala, Sweden).
data analysis
Stored raw data were transferred from Braided File Format to a
format compatible with a BioFocus integrator on an IBM PC computer.
Postrun analysis of the data was then done with the BioFocus 3000
software (version 3.0). The ssDNA sizes were determined by the linear
regression method to establish a curve of best fit for calibrators in
each series of analyses.
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Results and Discussion
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optimization of pcr and ce conditions
Nonspecific amplification of DNA is often observed on asymmetric
PCR, and nonspecific amplified products make data analysis difficult.
Because the sequence contains only adenosine repeats (Fig. 1
), the
ratio of dATP to other nucleotides (dGTP, dCTP, and dTTP) used was
10:1. By using the asymmetric PCR technique with dATP-rich nucleotides,
the ssDNA (30 nt) containing the primer (20 nt) and the
(A)10 repeat was amplified predominantly. The
polymerase probably does not continue beyond the poly(A) tract because
of the limiting concentrations of the other three nucleotides in the
reaction. The PCR products amplified with standard dNTP concentrations
gave various nonspecific peaks with molecular masses higher than the
target peak, but those amplified with dATP-rich nucleotides gave only a
specific peak (Fig. 2
). To obtain higher resolution, standard DNA was electrophoresed
at various voltages and temperatures; the higher the voltage, the
shorter the running time. However, the resolution was worse at higher
voltage. To analyze the 28–30 nt of the (A)10 repeat
rapidly, sufficient resolution was obtained at 500 V/cm within 30 min
(data not shown). Under these conditions, PCR products derived from
normal human DNA were subjected to CE analysis. To determine the
reproducibility of this method, the (A)10 repeat derived
from normal human DNA was amplified and was analyzed separately 10
times by CE. The CV of the found means was within 1.4% (data not
shown), indicating good reproducibility.
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Figure 2. Effects of the nucleotide concentrations on amplification
of the (A)10 repeat with the asymmetric PCR technique.
The electropherograms show the asymmetric PCR products amplified with
20 mmol/L dATP, 20 mmol/L dGTP, 20 mmol/L dCTP, and 20 mmol/L dTTP
(A), and 200 mmol/L dATP, 20 mmol/L dGTP, 20 mmol/L dCTP,
and 20 mmol/L dTTP (B). Other conditions were the same as
described previously (19). In each electropherogram, the
(A)10 repeat specific peak is indicated by an
arrow.
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resolution of this method
To confirm the resolution of this method, three kinds of DNA,
which contained the (A)9 repeat, (A)10
repeat, and (A)9 repeat mixed with the same amount of
(A)10 repeat, were subjected to CE analysis separately. The
number of adenines in the repeats was determined by a sequencing
technique used previously (data not shown). The (A)9 and
(A)10 repeat DNA showed one symmetric peak each, while the
(A)9 DNA mixed with (A)10 repeat DNA gave two
peaks in the electropherogram within 30 min (Fig. 3
) that were calibrated to be 29 nt and 30 nt, compared with
standard nucleotides. These data demonstrate that one nucleotide
difference could be detected with this method.
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Figure 3. Resolution of the amplified nucleotides using the
non-gel-sieving CE system.
The electropherograms show the (A)9 repeat DNA
(A), the (A)10 repeat DNA (B), and
the (A)9 repeat DNA mixed with the same amount of the
(A)10 repeat DNA (C). The capillary
electrophoretic conditions were given in Materials and
Methods.
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detection limit of this method
To evaluate the sensitivity of (A)10 repetitive
sequence analysis by CE, the PCR product containing (A)9
was mixed in twofold serial dilutions with the PCR product containing
(A)10. The mixtures were analyzed by non-gel-sieving CE.
When a sample composed of (A)9 and (A)10 in a
ratio of 0.125 or higher was subjected to this method, the peak derived
from (A)9 was detected in the electropherogram (Fig. 4
). Even at the lower DNA concentration, the peak derived from
(A)9 or (A)10 could be determined by comparison
with standard nucleotides described in Materials and Methods
(data not shown). These data indicate that this technique can be
applied for analysis of the clinical specimens containing 12.5%
deleted allele.
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Figure 4. Detection limit study on this system.
The (A)9 repeat DNA in twofold serial dilutions was
mixed with the (A)10 repeat DNA. The mixtures were
subjected to non-gel-sieving CE as described in Materials and
Methods. The ratios of the (A)9 repeat DNA to the
(A)10 repeat DNA are given above the electropherograms.
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analysis of the (a)10 repeat in colorectal cancers
On analysis of DNA derived from normal mucosa of a colorectal
cancer patient by non-gel-sieving CE, a 30-nt peak was found in the
electropherogram, whereas for the tumor DNA from the same patient an
additional 29-nt peak different from that of the normal DNA was
detected within 30 min (Fig. 5
). The additional peak was derived from the deletion of the
(A)10 repeat in TGF-ß RII. To analyze the
(A)10 repeat in TGF-ß RII, the conventional
method that contained SSCP analysis of PCR products followed by
sequencing was done (14). Nonspecific bands were sometimes
detected with the conventional method but not with the new method,
indicating that DNA amplification was done more specifically.
Therefore, this method may replace the conventional method. Moreover,
it can be applied to the analysis of DNA derived from paraffin-embedded
samples (data not shown), because the sizes of PCR products were much
shorter (28–30 nt).
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Figure 5. Detection of the deleted (A)10 repeat of
theTGF-ß RII gene in colorectal cancers.
The electropherograms are for normal DNA (N) and tumor DNA
(T) from the same patient.
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advantages
These data demonstrate that the amplified (A)10
repetitive sequence of TGF-ß RII using previously isolated
genomic DNA can be analyzed by non-gel-sieving CE within 30 min.
Whether specimens have a homozygotic or heterozygotic allele of the
(A)10 repeat can be shown more clearly by the number of
specific peaks in an electropherogram. The system involving
non-gel-sieving CE is performed automatically in a shorter time and can
be applied to the analysis of not only the (A)10 repeat but
also other microsatellite DNA containing two or three nucleotide
repeats. From the standpoint described above, this method is very
useful for routine microsatellite analysis of various diseases
including cancer.
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Acknowledgments
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We thank Mingde Zhu (Bio-Rad Labs.) for supplying us with the
polymer additives and Yoshimitsu Akiyama for supplying the cancer DNA.
This work was supported in part by a Grant-in-Aid for Developmental
Scientific Research from the Ministry of Education, Science, Sports and
Culture.
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Footnotes
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1 Nonstandard abbreviations: TGF-ß, transforming growth factor-ß; CE, capillary electrophoresis; ds, double stranded; ss, single stranded; SSCP, single-strand conformation polymorphism; and nt, nucleotide(s). 
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Abstract
Introduction
