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Technical Briefs |
1
Dept. of Clin. Chem., Res. Lab. of Atherosclerosis Genetics, P.O. Box 2000, Tampere Univ. Hosp., and
2
Dept. of Med. Biochem., Tampere Univ. Med. School, FIN-33521, Tampere, Finland;
a author for correspondence: fax 358-3-247-5554;
e-mail bltele{at}uta.fi
The traditional method for DNA separation is slab gel electrophoresis. Because slab gel electrophoresis, which involves the casting, loading, running, and staining of the gel is labor-intensive and time-consuming, high-resolution capillary electrophoresis (CE) has become an attractive alternative. CE, by contrast to conventional electrophoretic techniques, is capable of rapid, automated, reproducible, and high-resolution separation of minute amounts of DNA samples. To separate DNA fragments by CE, polyacrylamide gels or liquid buffers containing soluble polymers are used. Soluble polymers, such as hydroxyethyl cellulose, hydroxypropylmethyl cellulose, and methyl cellulose act as effective molecular sieves and allow for separation of DNA according to size (1). CE has recently shown promising results for the analysis of double-stranded DNA such as restriction fragment length polymorphisms and polymerase chain reaction (PCR) products (2)(3).
The insertion (I)/deletion (D) of a 287-bp sequence polymorphism within intron 16 of the gene for angiotensin-converting enzyme (ACE) is strongly associated with serum ACE concentrations (4). The D allele has been identified as a risk factor for the development of coronary heart disease and myocardial infarction (5). In addition, the deletion polymorphism has been associated with either microalbuminuria or overt nephropathy in diabetic patients (6)(7). Here we report a sensitive, simple, rapid, and nonisotopic procedure for identification of ACE I/D polymorphism by CE.
Amplification of genomic DNA ACE I/D polymorphism PCR products of a 190-bp fragment in the absence of the insertion and a 490-bp fragment in the presence of the insertion were generated. The sense oligonucleotide primer was 5'-CTG GAG ACC ACT CCC ATC CTT TCT-3', and the antisense primer was 5'-GAT GTG GCC ATC ACA TTC GTC AGA T-3' (8). The PCR mixture contained 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mL/L Triton X-100, 200 µmol/L each of the four deoxynucleotides, 1 µmol/L each of the primers, and 3 U of Dynazyme (Finnzymes) in a final volume of 50 µL. To enhance genotyping and to prevent misclassification of heterozygous individuals, we added 50 mL/L dimethyl sulfoxide to the reaction mixture (9). Blank controls, containing no genomic DNA, and positive controls (ID) were also run with each set of amplification. The amplification cycle was performed on a PTC-100 thermal cycler (MJ Research). After an initial denaturation at 96 °C for 3 min, the DNA was amplified by 30 PCR cycles—each consisting of denaturation at 94 °C for 1 min, annealing at 65 °C for 1 min, and extension at 72 °C for 2 min—followed by a final extension at 72 °C for 5 min. PCR products were directly analyzed by CE without being desalted or cleaned up.
CE separations were carried out with the use of the HP3D Capillary Electrophoresis System (Hewlett-Packard). CEP-coated fused-silica capillaries provided with the pGEM Double Stranded DNA Analysis Kit (Hewlett-Packard) of 56 cm effective length and 64.5 cm total length were used. The separation/flush buffer consisted of 89 mmol/L Tris, 332 mmol/L boric acid, and 2 mmol/L EDTA at pH 7.4 with additives of 15 g/L hydroxyethyl cellulose (Hewlett-Packard). The buffer was prepared according to the manufacturer's recommendation, filtered, and degassed by sonication. The cathode was set on the injection side, the anode on the detection side. Samples were introduced into the capillary by electrokinetic (-10 kV for 30 s) injection, and the separation was conducted at constant voltage of -22 kV (340 V/cm). The capillary was held at 20 °C throughout the experiment, and ultraviolet absorbance was monitored at 258 nm. Before each injection, a 10-min buffer wash was performed to ensure column cleanliness and reproducibility. A typical assay of samples lasted for 30 min. The identification of the various DNA fragments by size was obtained by plotting log bp vs 1/migration time.
Fig. 1
A shows an electropherogram of the separation of pGEM DNA
calibrator with the use of a 15 g/L hydroxyethyl cellulose-filled
capillary at -22 kV. The separation conditions were optimized in
several preliminary experiments with electrokinetic and pressure
injection; electrokinetic injection gives higher resolution. The
separation conditions in Fig. 1A
were sufficient for the rapid and
complete separation of the pGEM sample, which contained 15 fragments
ranging from 36 to 2645 bp. The DNA calibrator was run before the
samples to ensure proper calibration of the analysis. Fig. 1B
shows an
example of a sample from a heterozygous individual, who expresses both
490- and 190-bp fragments. To confirm the accurate identification of
the ACE gene polymorphism, we ran 10 samples, and all genotypes
determined by CE were consistent with the results obtained by agarose
gel electrophoresis.
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We have demonstrated excellent separation efficiency for PCR products of the ACE gene by CE, in substantially shorter analysis time than by slab gel electrophoresis. Several investigators have recently reported that PCR analysis by CE may be hindered by salt in the reaction mixture, so salt must be removed before the CE analysis (10). However, in the present study the ACE gene PCR products were analyzed directly by CE without further sample treatment, which saves time and resources. In addition, samples can be loaded onto an autosampler and run overnight, unattended, and analyzed data are directly entered into computer. In our studies, one coated capillary column lasted for >1 month and was used for hundreds of assays. Furthermore, this method avoids the use of hazardous materials such as ethidium bromide or radioactivity. Taken together, CE provides a simple, rapid, sensitive, and accurate method for the identification of the ACE I/D gene polymorphism.
Acknowledgments
This work was supported by grants from the Finnish Foundation of Cardiovascular Research, Medical Research Fund of Tampere University Hospital, and the Elli and Elvi Oksanen Fund of the Pirkanmaan Regional Fund under the auspices of the Finnish Cultural Foundation.
References
The following articles in journals at HighWire Press have cited this article:
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  R. Lehmann and M. Koch Capillary electrophoresis in the analysis of the deletion/insertion polymorphism of the angiotensin I-converting enzyme gene. Clin. Chem., July 1, 1998; 44(7): 1582 - 1583. [Full Text]
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