Establishment And Application Of3-D Hydrogel Microarray For SNP Detection Based On Specific Probe Extension

A single nucleotide polymorphism is a genomic DNA sequence polymorphism caused by a single nucleotide variation. As the third-generation genetic marker, SNP determine the susceptibility to diseases and the sensitivity to medical treatment of different population. Hence, SNP detection has a remarkable influence on early diagnosis and reasonable medication. Current SNP detection methods include restriction fragment length polymorphism(RFLP), single strand conformation polymorphism(SSCP), allele-specific polymerase chain reaction(AS-PCR), sequencing, denaturing high-performance liquid chromatography(DHPLC), etc. However, the methods mentioned above are all laborious, time-consuming, low-throughput and demanding expensive equipments and reagents. Moreover, these can only be operated in laboratory, hardly fulfilling clinical demands. DNA chip is a method that can obtain vast SNP information on one chip, featuring rapidness, high-sensitivity and high-throughput. However, traditional two-dimensional hybridize DNA chip has such shortages as low-efficiency of immobilization and hybridization, thus it costs a long time in hybridization and has poor ability to recognize base mismatches, producing relatively high rate of false-positive results.To address the problems of two-dimensional DNA chip, polyacrylamide gel is introduced to gene chip construction. Polyacrylamide gel is characterized by its unique three-dimensional porous conformation, contributing to large volume for nucleotide immobilization, which increase the sensitivity of detection. Moreover, polyacrylamide gel can offer an atmosphere resembling liquid reaction environment, which is favorable to hybridization. However, this method is precious because each SNP requires a pair of specific fluorescence-labeled probes, restricting its large-scale application in clinic and research.To address the problem, a novel SNP genotyping chip was established based on three dimensional polyacrylamide gel, featuring high-throughput, low-cost and universality. This method utilized non-fluorescent probe coupled with Cy5-dCTP extension to label target DNA, circumventing the use of fluorescent probe, then achieving the universality of fluorescent labeling with low cost. Meanwhile, electrophoresis could remove the Cy5-dCTPs absorbed in gel and the probes hybridized non-specifically, reducing background signals and false-positive signals. In addition, our method had a low risk of cross-contamination from PCR amplicons due to no need of purification step for PCR products.This paper investigated PCR reactions, the feasibility of extension reactions in gel, DNA polymerase selection and the clearance conditions hybridized probes, established a SNP chip platform based on three dimensional polyacrylamide gel. This method is applied to40clinical samples detecting14417G>C (rs11053646). The results are in accordance with pyrosequencing results.Overall, the SNP genotyping chip based on three-dimensional polyacrylamide gel gives rise to a new platform and tool for life science research and clinical investigation, shading light on pharmacogenomics and early diagnosis.