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Technical Briefs |
1 Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences, 2 Frontier Center for Microbiology, and 3 Department of Medicine and Molecular Science, Division of Frontier Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan;
aaddress correspondence to this author at: Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan; fax 81-82-257-5336, e-mail kinoeiji{at}hiroshima-u.ac.jp
Various procedures have been developed (1)(2)(3)(4) to facilitate high-throughput single-nucleotide polymorphism (SNP) detection for establishment of genetic linkage and aiding in diagnosis and treatment of inherited diseases. Such procedures, however, usually require expensive equipment and skilled analysts, making it very difficult for most clinical researchers and physicians to obtain useful SNP data. Thus, the establishment of a reliable and cost-effective SNP detection method that uses general equipment is desirable.
We developed a simple, rapid, cost-effective, and accurate method for the detection of mutations by polyacrylamide gel electrophoresis with the additive Zn2+–1,4,7,10-tetraazacyclododecane (cyclen) complex (Rf Enhancer-ZC; Toyobo), called Zn2+–cyclen–PAGE (5). The combination of a PCR-based heteroduplex method and Zn2+–cyclen–PAGE enables accurate detection of single mutations introduced artificially, even for less detectable substitutions such as A-T to T-A and G-C to C-G. This procedure does not require radioisotopes or fluorescent probes. Heteroduplex bands that arise from annealing of complementary strands, one from mutant and one from wild-type DNA (heterozygosity) are identified in the electrophoresis gel during PCR.
Our approach is based on 3 principles: (a) a single-base mismatch produces a local conformational change in the double-stranded DNA, leading to differential migration of the heteroduplex and homoduplex bands; (b) the addition to the gel of Zn2+–cyclen, which selectively binds to the thymine base (T) and disrupts the double strands, intensifies the local conformational change, increasing differential migration of both duplexes; and (c) binding of Zn2+–cyclen to T decreases the total charge of the target DNA, thus enhancing detection. Slow or differentially migrating bands in the gel indicate the presence of heteroduplex bands, which suggest the existence of a mutation or polymorphism. Furthermore, Zn2+–cyclen–PAGE separates homoduplexes of specific mutant alleles from homoduplexes of their homologous wild-type alleles. We achieved higher resolution of Zn2+–cyclen–PAGE by adopting a discontinuous buffer system with a separating gel and a stacking gel (6)(7)(8).
As the first practical use of the improved method, we analyzed heterozygosity in the human cardiac sodium channel gene, SCN5A. Many mutations in the SCN5A gene, which consists of 28 exons spanning 
80 kb on chromosome 3, are responsible for multiple arrhythmia disorders, including long QT syndrome type 3 (LQT3), idiopathic ventricular fibrillation (IVF), inherited cardiac conduction defects, and the Brugada syndrome. More than 80 mutations associated with the Brugada syndrome have been identified (9) since the first indication of a genetic basis in 1998(10). Because these mutations are scattered throughout the SCN5A gene, a comprehensive and accurate genomic analysis is required to confirm the classification of Brugada syndrome as an inherited disease, to predict the clinical phenotype, and to develop suitable therapies.
Eighteen unrelated Japanese individuals participated in this study: 10 patients with Brugada syndrome, 1 with IVF without ST segment elevation in the right precordial electrocardiogram leads, 1 with LQT3, and 6 healthy individuals without family histories of syncope or sudden death. All disease diagnosis was performed at Hiroshima University Hospital. The study protocol was approved by the human genome research ethics screening committee of Hiroshima University, and written informed consent for participation was obtained from all participants. Peripheral blood (10 mL) was obtained from each participant, and genomic DNA was extracted from the leukocytes according to a standard protocol by use of the QIAamp DNA Blood Maxi Kit (Qiagen). All SCN5A exons and those splice sites were amplified by PCR from 2.5 ng of genomic DNA with KOD-plus or Blend Taq-plus DNA polymerase (Toyobo). The untranslated region of exon 28 was not analyzed in this study. The PCR procedure was described previously (11). The optimum primers used (98 pairs; primer sets are listed in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue11) were originally designed according to the sequences of GenBank accession nos. AP006241 (chromosome 3) and M77235 (cDNA of SCN5A) so that, for accurate mutation detection, the length of each PCR product would not exceed 200 bp. The primers were purchased from Texas Genomics Japan and Espec Oligo Service.
The modified Zn2+–cyclen–PAGE was performed at 25 mA for 100 min at room temperature in a 1-mm-thick, 9-cm-wide, and 9-cm long gel on a standard minislab PAGE apparatus (Model AE6500; ATTO). The gel consisted of 1.8 mL of stacking gel (45 g/L polyacrylamide and 125 mmol/L Tris-HCl, pH 6.8) and 6.3 mL of a separating gel (5.0 mmol/L Zn2+–cyclen, 200 g/L polyacrylamide, and 375 mmol/L Tris-HCl, pH 8.8). The acrylamide stock solution was prepared as a mixture of a 99:1 ratio of acrylamide to N,N'-methylenebisacrylamide. The cathode buffer was 25 mmol/L Tris and 192 mmol/L glycine, and the anode buffer was 25 mmol/L Tris and 192 mmol/L glycine containing 5.0 mmol/L Zn(NO3)2. Each PCR product for Zn2+–cyclen–PAGE was dissolved in a half amount of a loading dye containing 50 mmol/L EDTA, 0.5 g/L bromphenol blue, and 300 mL/L glycerol and then applied (0.5–1.0 ng of DNA per well). The DNA bands were visualized by staining with 10 000-fold–diluted SYBR Green I (15 mL per gel; Cambrex Bio Science Rockland) after electrophoresis. The entire amount of SCN5A gene (i.e., 41 DNA fragments x 12 patients) and the DNA fragments, including the samples showing multiple bands (healthy individuals nos. 14 and 16), were sequenced with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The primers (41 pairs) used for direct sequencing were the same as those reported previously (12). Direct sequencing revealed that all patients had the SCN5A coding the same hH1c channel isoform(13), entered as GenBank accession no. AY148488.
This novel screening method disclosed that 9 patients and 2 healthy individuals had various heterozygosities in the SCN5A gene. Examples of typical Zn2+–cyclen–PAGE results are shown in Fig. 1
. The higher resolution achieved with the same PCR products from exon 10 for electrophoresis with a discontinuous buffer system is apparent in Fig. 1A
compared with Fig. 1B
[continuous buffer system was 90 mmol/L Tris and 90 mmol/L borate (5)]. Two additional differentially migrating bands representing heteroduplexes (lane 1 of Fig. 1A
) were confirmed more clearly by use of the newly modified Zn2+–cyclen–PAGE. Subsequent direct sequencing of the DNA fragment including exon 10-2 of patient 1 revealed a heterozygous nucleotide substitution (G1212A) that does not affect codon L404. A similar synonymous SNP (i.e., G87A in codon A29) was also detected in exon 2 of 5 Brugada syndrome patients and 2 healthy individuals (Fig. 1C
). We also detected a heteroduplex (lane 2 of Fig. 1D
), which we identified as a heterozygous G-to-A transversion in the 5' splice junction of the intron between exons 21 and 22, suggesting abnormal splicing linked to the Brugada syndrome. No nucleotide change affecting the coding was detected in the other Brugada patients. In some previous studies, the reported incidence of SCN5A mutations in Japanese patients with the Brugada syndrome varied from 2% to 27%(14); therefore, further genetic testing will be required to understand the genotype–phenotype relationship in this disease. Moreover, Zn2+–cyclen–PAGE showed another abnormality in exon 20 of IVF and LQT3 patients (lanes 11 and 12 in Fig. 1E
) attributable to a common nucleotide alteration (G3575A) leading to a codon mutation of R1192Q. This mutation has been reported to be associated with the Brugada(15) and LQT3(16) syndromes. It is interesting to detect the same SNP in an IVF patient without typical electrocardiographic signs of the Brugada syndrome. No heterozygous mutation associated with changes in the codon was detected in the control individuals. Direct sequencing disclosed no mutation in the patient samples showing a single DNA band, indicating that all heterozygous mutations in the SCN5A gene tested were detected by our screening. Because the samples showing a single DNA band in this study and a previous report(5) had no mutations, the detection sensitivity of our screening would not be less than that (generally 70%–90%) of other gel-based approaches such as single-strand conformation polymorphism analysis and denaturing gradient gel electrophoresis.
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In conclusion, our Zn2+–cyclen–PAGE method is a novel genetic approach to diagnosing autosomal dominant inherited diseases. Heteroduplexing with the homologous wild-type allele would also enable genotyping of recessive-mode diseases. Use of the discontinuous buffer system sharpened each DNA band in the minislab gel at room temperature, making it easier to reliably detect heteroduplex bands. Zn2+–cyclen–PAGE requires a general minislab PAGE system and an additive, Zn2+–cyclen, without any special apparatus. The cost of Zn2+–cyclen for 1 SCN5A gene (98 DNA fragments) was less than $4.00 US dollars. This method may therefore be feasible for initial screening of hereditary diseases in a clinical laboratory.
Acknowledgments
We sincerely thank the individuals who provided blood samples for this study. We also thank the Research Center for Molecular Medicine, Graduate School of Biomedical Sciences, Hiroshima University, for the use of their facilities. This work was supported by Grants-in-Aid for Scientific Research (B) (15390013) and for Young Scientists (B) (14770014) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References
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