Development and evaluation of thrombosis susceptibility gene chip / 吉林大学学报(医学版)

ABSTRACT

Objective: To explore the development methods and prelimnary clinical validation of thrombos susceptibility gene chip, and to illustrate a rapid and high-throughput method for detecting the thrombos susceptibility gene mutation. Methods: According to the published gene sequences of thrombotic susceptibility genes in GenBank, the reference probes and special probes were pointed on the glass slide. After ultraviolet crosslinking, thrombois susceptibility gene chip was eatablished. The validity of gene chip was tested by potive reference (mutant gene and normal gene at each detection site) and negative reference (distilled water) as templates, thereby performing PCR reaction. The specificity and sensitivity of gene chip were detected by using the human genome DNA of target sequence proven by sequencing as templates, thereby performng PCR reaction. Meanwhile 150 healthy subjects and 24 thrombosis patients with family hstory of unexplained thrombotic dsease from Jilin province, Henan provnce and Yunnan province were carried out the clinical verification of gene chip. The analyss index was the hybridization signal trength of the correspondng ite. Results: The hybridization results of postive reference and negative reference as templates showed that the specific hybridization signals appeared at the corresponding ites, which indicated that the detection sites of gene chip are effective. The gene chip used to detect the selected mutation sites had specific hybridization signals, suggesng there were good specificity of gene chip. The senstivity of gene chip was 50 - 100 mg · L-1 by testng genomic DNA with stepwise dilution. Eight individuals with thrombosis susceptibility gene mutations were found in 150 healthy subjects. Twenty ndividuals with thrombosis susceptibility gene mutations were found in 24 thrombosis patients with family hstory of unexplaned thrombotic dsease. The statistcs results showed that the detection rate of thrombosis susceptibility gene mutations in the thromboss patients with unexplaned thrombotic disease family history was significantly higher than that of the healthy subjects (P<0.05). Conclusion: The developed thrombosis susceptibility gene chip has great specificity, senstivity, and high detection rate of thromboss susceptibility genes. It has potental values in the early dagnosis and susceptibility sk assessment of thrombotic dseases.