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
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.
