title: A low-cost hydrogel chip for SNP typing by the incorporation of Cy5-dCTP into label-free allele-specific probes hybridizing to gel-immobilized targets.
abstract: A 3-dimensional (3-D) polyacrylamide gel microarray based on dual-color fluorescence hybridization was an efficient SNP typing method with a high-throughput, but it is expensive to use dual dye-labeled allele-specific probes to type various SNPs. To lower the typing cost on 3-D polyacrylamide gel microarray, we propose a novel method by incorporating Cy5-dCTP into label-free allele-specific probes hybridizing to gel-immobilized targets. The method is much simple. At first, raw PCR products without any purification was spotted on the acryl-modified slides to copolymerize with acrylamide monomers. Then a pair of allele-specific probes were respectively added into two different areas of a hydrogel chip to hybridize with the single-stranded DNA targets immobilized in the gel-pads. Before extension reaction with Cy5-dCTP, electrophoresis was performed on the gel chip to remove non-specific allele-specific probes, and a high specificity was thus obtained. After the extension reaction, electrophoresis was used once more to remove the unincorporated Cy5-dCTP absorbed in the gel pads, and a low background image was achieved. The method was successfully employed to type the SNP (C14417G) in the OLR-1 gene for 40 different samples, and the typing results were consistent with those by pyrosequencing, indicating that the proposed method is accurate and specific in SNP typing. As no use of dye-modified probes, the typing cost is significantly decreased in comparison with the conventional typing method based on dual-color fluorescence hybridization, in particular when typing multiple SNPs. In addition to the low cost, our method has a low risk of cross-contamination from PCR amplicons due to no need of purification step of PCR products. Although only proof-of-concept results were given, we believe that the proposed method should be very useful for screening the biomarkers related to disease-susceptibility and personalized medicine where detection of many SNPs in different genes is required.
authors.affiliation: School of Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Zhiyao
authors.initials: Z
authors.lastname: Chen
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Fu
authors.forename: Youzhi
authors.initials: Y
authors.lastname: Li
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Tang
authors.forename: Haiping
authors.initials: H
authors.lastname: Wu
authors.forename: Bingjie
authors.initials: B
authors.lastname: Zou
authors.forename: Guohua
authors.initials: G
authors.lastname: Zhou
mesh_terms: D000483:Alleles; D002232:Carbocyanines / Q000378:metabolism*; D015342:DNA Probes; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D020100:Hydrogels; D020641:Polymorphism, Single Nucleotide
chemical_list: D002232:Carbocyanines; C544355:Cy5-dCTP; D015342:DNA Probes; D003845:Deoxycytosine Nucleotides; D020100:Hydrogels