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.
文摘目的:胸腺肽β10(thymosinβ10,TMSβ10)在部分恶性肿瘤中呈过表达状态,但其在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中的表达尚无报道。本研究旨在探讨TMSβ10在OSCC组织中的表达和潜在的作用机制。方法:收集OSCC样本基因芯片和RNA测序表达矩阵,提取TMSβ10 mRNA表达值;采用标准化均值差(standardized mean difference,SMD)总受试者工作特征(summary receiver operating characteristic,SROC)曲线评估TMSβ10表达的临床价值,并进行TMSβ10相关基因的功能富集分析,再以免疫组织化学实验进行验证。结果:与252份非癌口腔组织相比,TMSβ10在677例OSCC组织中显著升高[SMD=1.68(0.95~2.42)],且对于OSCC具有高区分能力[SROC曲线下面积(area under the curve,AUC)=0.95,95%CI为0.92~0.96]。TMSβ10相关基因生物学功能主要富集于伤口愈合、蛋白酶体蛋白等方面。此外,多个数据集的分析显示纤维连接蛋白1(fibronectin 1,FN1)与TMSβ10基因之间存在显著的正相关性(r为0.34~0.83,均P<0.05),被鉴定为TMSβ10伤口愈合通路中的枢纽基因。结论:TMSβ10表达上调可能具有促进OSCC发生的作用。
