Objective To screen coronaryartery disease (CAD) specific expressions and clone their genes. Method Blood samples were collected from CAD and non - CAD patients at the end of coronary angiography. mRNA from samples wa...Objective To screen coronaryartery disease (CAD) specific expressions and clone their genes. Method Blood samples were collected from CAD and non - CAD patients at the end of coronary angiography. mRNA from samples was isolated and converted into cDNA. After ligated with specific linkers, the cDNA was amplified with complementary primers. PCR products from CAD samples were named as tester; the ones from non - CAD samples were named as driver. With different ratio of tester to driver (1 : 100,1: 1, 000, and 1: 10, 000), they were mixed, denatured, and renatured. Single strand cD-NA was eliminated by Mung bean nuclease. Double strand cDNA presented only in tester was amplified, ligated in vector pUC19 and pUC53, and transformed into E. coll DH5a. Strains with inserted cDNA fragments were picked up based on blue and white selection. Insertions were screened by endonuclease digestion and DNA sequencing. Results were compared with DNA sequences of GeneBank. Results: After the selection with representational differential analysis, CAD specific cDNA fragments with different sizes (about 1kb, 0. 75kb, and 0. 6kb) were cloned. Among them, two fragments from unknown genes were identified. One presented a 43. 3 % similarity with part of the rattus norvegicus lipocortin gene. Another presented a 45. 4 % similarity with part of the human polynucleotide kinase 3' - phosphatase gene. Conclusion There are at least two CAD specific - ex- pressions from unknown genes that were partially similar to lipocortin and polynucleotide kinase 3'- phos-phatase genes, respectively. Expression of these genes might affect the formation and progression of plaque within coronary artery.展开更多
提出了一种检测三磷酸腺苷(ATP)的非标记荧光新方法,该方法基于T4多聚核苷酸激酶调控Lambda核酸外切酶活性的原理,利用DNA荧光染料SYBR Green I作信号报告基团.有ATP时,双链DNA底物能在T4 PNK作用下发生5'端磷酸化,进而被核酸外切...提出了一种检测三磷酸腺苷(ATP)的非标记荧光新方法,该方法基于T4多聚核苷酸激酶调控Lambda核酸外切酶活性的原理,利用DNA荧光染料SYBR Green I作信号报告基团.有ATP时,双链DNA底物能在T4 PNK作用下发生5'端磷酸化,进而被核酸外切酶降解成DNA碎片,得到较弱的荧光信号,信号强度与ATP浓度成反比.该方法对ATP检测的线性范围为0.04~4 mmol/L,检测限为20 nmol/L.实验结果表明本方法具有快速、成本低、灵敏度高和简单易操作等优点,可进一步应用于复杂生物样品的分析.展开更多
文摘Objective To screen coronaryartery disease (CAD) specific expressions and clone their genes. Method Blood samples were collected from CAD and non - CAD patients at the end of coronary angiography. mRNA from samples was isolated and converted into cDNA. After ligated with specific linkers, the cDNA was amplified with complementary primers. PCR products from CAD samples were named as tester; the ones from non - CAD samples were named as driver. With different ratio of tester to driver (1 : 100,1: 1, 000, and 1: 10, 000), they were mixed, denatured, and renatured. Single strand cD-NA was eliminated by Mung bean nuclease. Double strand cDNA presented only in tester was amplified, ligated in vector pUC19 and pUC53, and transformed into E. coll DH5a. Strains with inserted cDNA fragments were picked up based on blue and white selection. Insertions were screened by endonuclease digestion and DNA sequencing. Results were compared with DNA sequences of GeneBank. Results: After the selection with representational differential analysis, CAD specific cDNA fragments with different sizes (about 1kb, 0. 75kb, and 0. 6kb) were cloned. Among them, two fragments from unknown genes were identified. One presented a 43. 3 % similarity with part of the rattus norvegicus lipocortin gene. Another presented a 45. 4 % similarity with part of the human polynucleotide kinase 3' - phosphatase gene. Conclusion There are at least two CAD specific - ex- pressions from unknown genes that were partially similar to lipocortin and polynucleotide kinase 3'- phos-phatase genes, respectively. Expression of these genes might affect the formation and progression of plaque within coronary artery.
