Mutations in cardiac troponin I （cTnI） gene were assessed based on gene-chip technology.Special probes were designed to fabricate the low-density gene-chip,which could detect the mutations in exons 3,5,7,and 8 of th...Mutations in cardiac troponin I （cTnI） gene were assessed based on gene-chip technology.Special probes were designed to fabricate the low-density gene-chip,which could detect the mutations in exons 3,5,7,and 8 of the cTnI gene simultaneously.For each exon,two oligonucleotide sequences labeled with fluorescein at the 5＇-end were designed,one （oligonucleotide Ⅰ） simulating the wild type and the other （oligonucleotide Ⅱ） simulating the mutant.Oligonucleotides Ⅰ and Ⅱ were mixed together to simulate the heterozygote.After optimizing the hybridization protocols,the fabricated gene-chip could detect the mutations in the exons of the cTnI gene with relative high sensitivity and specificity.The fully complementary probe gave a fluorescent signal almost 50% stronger than that of the one-base mismatched one,which is in accordance with the result from a theoretical estimate. An applicable special gene-chip is available to investigate and diagnose familial hypertrophic cardiomyopathy （FHCM） after further improvement.展开更多
Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (...Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.展开更多
基金The National Natural Science Foundation of China(No.60071001)China Postdoctoral Science Foundation(No.2002)+2 种基金Trans-Century Training Programme Foundation for the Talents by theState Education Commission(No.[2004]527)the Foundation of the 135?Key Laboratory of Jiangsu Province(No.SK200205)the HighTechnology Research Plan of Jiangsu Province(No.BG2003033,BG2001010).
文摘Mutations in cardiac troponin I （cTnI） gene were assessed based on gene-chip technology.Special probes were designed to fabricate the low-density gene-chip,which could detect the mutations in exons 3,5,7,and 8 of the cTnI gene simultaneously.For each exon,two oligonucleotide sequences labeled with fluorescein at the 5＇-end were designed,one （oligonucleotide Ⅰ） simulating the wild type and the other （oligonucleotide Ⅱ） simulating the mutant.Oligonucleotides Ⅰ and Ⅱ were mixed together to simulate the heterozygote.After optimizing the hybridization protocols,the fabricated gene-chip could detect the mutations in the exons of the cTnI gene with relative high sensitivity and specificity.The fully complementary probe gave a fluorescent signal almost 50% stronger than that of the one-base mismatched one,which is in accordance with the result from a theoretical estimate. An applicable special gene-chip is available to investigate and diagnose familial hypertrophic cardiomyopathy （FHCM） after further improvement.
文摘Hypertrophic cardiomyopathy (HCM) is one of the diseases damaging people health most badly and some mutations of exons in cardiac troponin I (cTnI) gene are closely associated with family hypertrophic cardiomyopathy (FHCM).A microarray was fabricated to screen mutations in exons 3,5,7,and 8 in cTnI gene.Primers were designed for the PCR (polymerase chain reaction) to amplify the target DNA fragments from fresh blood samples.In order to simplify the PCR process,multiplex PCR technology was investigated in detail.The concentration of Mg^(2+) played an important role in multiplex PCR process,a properly low concentration of Mg^(2+) submitted a better speciality of PCR products.The speciality was also favored when the annealing temperature was reasonably enhanced and 64℃is the optimal annealing temperature for the multiplex PCR systems.When applying the fabricated gene-chip to detect the target fragments from PCR mixture,the signal intensity sequence is in accordance with that from theoretic estimate.
