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Abstracts of Oak Ridge Posters |
1 Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
aauthor for correspondence: fax 86-25-83619983, e-mail zhlu{at}seu.edu.cn
Robust, inexpensive, high-throughput methodologies are needed for the analysis of molecular markers. DNA microarrays are widely used in biological and biomedical research as a high-throughput tool. At present, there are two general microarray-based methods for genotyping. One approach involves arraying thousands of short oligonucleotides on glass slides for detection of many single-nucleotide polymorphism (SNP) loci in target DNA (1). This method is particularly well suited for genotyping thousands of markers in a limited number of individuals (2)(3)(4)(5)(6). The other approach involves arraying amplified PCR products on glass slides to detect a few SNPs in a large number of samples. Recently, Flavell et al. (7) developed a tagged microarray marker approach for scoring thousands of samples for a codominant molecular marker. Biotin-terminated allele-specific PCR products are spotted unpurified on streptavidin-coated glass slides and visualized by hybridization of fluorescent detector oligonucleotides to tags attached to the allele-specific PCR primers. The tagged microarray marker approach is an efficient method for genotyping thousands of samples on a glass slide. However, reliable allele-specific PCR conditions are needed. Moreover, the expensive streptavidin-coated glass slides can be stored for only 3 days in phosphate-buffered saline.
For many applications in the field of biological research, PCR products are the more convenient probe molecules (8). We have developed a microarray-based method, based on dual-color fluorescence hybridization, for genotyping of SNPs in thousands of individuals (9). The experiment successfully demonstrated that PCR products subjected to dual-color hybridization on a microarray could used to analyze molecular markers. However, two fluorescently labeled detector probes are required for each SNP, and the cost is high when many different SNPs need to be analyzed.
In this report, we describe a high-throughput method for genotyping of any SNP from hundreds of individuals by use of two pairs of secondary labeled universal oligonucleotides (detectors).
Peripheral blood was obtained from 196 patients suffering from different types of hematologic malignancies. All were diagnosed cases referred from ZhongDa Hospital and The First Affiliated Hospital of Nanjing Medical University (Nanjing, China). Genomic DNA was isolated by standard methods using proteinase K digestion and phenol–chloroform extraction. The genotype of the human mismatch repair gene hMLH1 at the G93A locus was amplified by PCR with 0.4 µmol/L each of the forward (5'-AGTAGCCGCTTCAGGGA-3') and reverse (5'-AGGACGGTGCGGTGAGAGTG-3') primers, as described previously (10). The profile consisted of an initial melting step of 4 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C; with a final elongation step of 7 min at 94 °C. PCR mixtures contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 200 µM each of the deoxynucleotide triphosphates, and 1.25 U of Taq DNA polymerase (TaKaRa) in 50 µL. PCR was preformed in a PTC-225 thermocycler (MJ Research).
PCR products were purified by use of a QIAquick column (Qiagen). The eluted PCR products were dried under reduced pressure and resuspended in 3x standard saline citrate (SSC). Each sample was then diluted with 3x SSC supplemented with 1.5 mol/L betaine to a final concentration of 200 µg/L and transferred to new 384-well plates for arraying. After printing, the microarrays were dehydrated for 30 s in a humid chamber and then snap-dried for 2 s on a hot plate (100 °C). The DNA was then cross-linked to the surface by subjecting the slides to 60 mJ of energy (Stratagene Stratalinker) and baking them for 2 h at 80 °C. The rest of the poly-L-lysine surface was blocked by incubation for 15 min in a solution consisting of 1-methyl-2-pyrrolidinone (Aldrich) and 1 mol/L boric acid (pH 8.0). Directly after the blocking reaction, the bound DNA was denatured by incubation for 2 min in distilled water at 95 °C. The slides were then transferred to a bath containing 950 mL/L ethanol at room temperature, rinsed, and then spun dry in a clinical centrifuge. Slides were stored in a closed box at room temperature until used.
The sequences for the probes and fluorescent detectors (Shenyou Inc.) used for genotyping the hMLH1 G93A polymorphism are shown in Table 1
. Probes 93G and 93A were mixed in equimolar amounts and suspended in unihybridization solution (3:1 dilution by volume; Telechem). Hybridization was conducted in a moist hybridization chamber under a cover slip at 37 °C for 4 h. After hybridization, the slides were rinsed and then washed successively at room temperature with 2x SSC containing 1 g/L sodium dodecyl sulfate, 0.1x SSC containing 1 g/L sodium dodecyl sulfate, and deionized water (5 min for each wash), and dried by centrifugation at 38g for 5 min. Detectors A-B, A'-B', C-D, and C'-D' were mixed and hybridized as described above. The scheme for SNP genotyping is illustrated in Fig. 1A
.
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In principle, probe hybridization should produce different spot colors depending on the SNP genotype, as shown in Fig. 1A
. The homozygous wild type yielded strongly fluorescent Cy3 spots (green fluorescence). A strongly fluorescent Cy5 spot (red fluorescence) was produced by the homozygous mutant. The heterozygote yielded both Cy3 and Cy5 spots; because of overlapping, the fluorescence was strongly "yellow".
The results obtained for 196 genomic DNA samples from patients with hematologic malignancies analyzed for the G93A locus are shown in Fig. 1B
. The complete array was spotted in quadruplicate, and the fluorescence pattern was highly reproducible between replicates (Fig. 1B
). Spot homogeneity was dependent on the variation in the DNA concentration across a spot. Supplementing SSC with 1.5 mol/L betaine yielded much more homogeneous spots (11).
The fluorescence signals from seven representative samples were quantified (Fig. 1C
). Homozygous samples had a fluorescence ratio of 
10 (stronger:weaker signal), whereas heterozygous samples had ratios near 1.0. The microarray results were additionally validated by sequencing (data not shown).
The method described here requires only simple PCR and scoring. The set-up involves simple PCR and scoring by microarray scanning, which has been well developed for expression screening and takes only a few minutes to carry out. Just as for conventional expression microarrays, the dual-color hybridization approach yields robust data if replicate experiments are performed, with the PCR products printed multiple times in each experiment. One major advantage of this method is that the cost is very competitive. Two pairs of universal labeled oligonucleotides can be used for genotyping any one SNP locus in thousands of individuals.
In summary, our experiments successfully demonstrated that the microarray-based method with layer-by-layer dual-color hybridization could be applied as an inexpensive and high-throughput tool for SNP genotyping in a large number of individuals.
Acknowledgments
The National Key Fundamental Research Foundation, the National Natural Science Foundation of China, and the National High-Tech Research Program supported this work.
References
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