Association Study of NOD2 Gene and Coronary Artery Disease Based on Optimized DNA Ligase Chain Reaction / 中国循环杂志

ABSTRACT

Objective: Based on optimized method of DNA ligase chain reaction in medium/low throughput genotyping, we assessed the relationship between NOD-like receptor genes NOD1, NOD2 and coronary artery disease (CAD) occurrence. Methods: A multiplex PCR was conducted to enrich DNA template; probe design, annealing temperature, time and number of circulation of PCR were opfimizecl for allele specific ligation; allele specific products were identified by fluorescence PCR and capillary electrophoresis; the accuracy was verified by Sanger sequencing. Single nucleotide polymorphisms (SNPs) on NOD1 gene and NOD2 gene were examined in 1555 CAD patients and 1887 control subjects; the relationship between SNPs and CAD occurrence was studied. Results: Based on optimized PCR condition and allele specific probe design, 30 allele loci genotyping can be genotyped by 10ng DNA template at one time. Association study presented that rs751271 and rs1861759 on NOD2 gene were related to non-hypertensive CAD, allP<0.05; with Bonferroni correction, such correlation was still significant, allP<0.05. Conclusion: We optimized DNA ligase chain reaction and established a novel high accuracy, low cost method for the demand of medium/low throughput genotyping in clinical molecular diagnosis. With this method, we identified that rs1861759 and rs751271 on NOD2 gene were associated with non-hypertensive CAD.