A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip ...A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip is simulated and analyzed.The optimal size of the chip is 30×22 mm2, the roundabout micro-channel is the 90 μm width, 40 μm depth. Two micro-heater with the nickel-chrome alloy material film are formed on the side of silicon belonging to denaturation and renaturation zones needed for PCR reaction, and two adiabatic structures with groove on side of silicon by anisotropy etching. By the mode of heating local zones at single side, three wider constant temperature zones could be formed, which are 60 ℃,72 ℃,95 ℃ and suitable for PCR,and the temperature-difference could be restricted in less than 5 ℃.展开更多
基金the National Natural Science Foundation of China(Grant No.60576047)
文摘A novel continuous-flow PCR chip adopting self-heating, passive-cooling mode to realize the DNA fragments amplification was presented. Using the ANSYS finite element analysis, the temperature distribution of the chip is simulated and analyzed.The optimal size of the chip is 30×22 mm2, the roundabout micro-channel is the 90 μm width, 40 μm depth. Two micro-heater with the nickel-chrome alloy material film are formed on the side of silicon belonging to denaturation and renaturation zones needed for PCR reaction, and two adiabatic structures with groove on side of silicon by anisotropy etching. By the mode of heating local zones at single side, three wider constant temperature zones could be formed, which are 60 ℃,72 ℃,95 ℃ and suitable for PCR,and the temperature-difference could be restricted in less than 5 ℃.
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
文摘为建立一种高同源区段的单核苷酸多态性(SNP)基因分型技术,通过构建本地Blast对SNP所在的200和400 bp区段进行同源性评估,并筛选出高同源区段的SNP。利用第一轮多重长PCR(polymerase chain reaction)捕获329个样本的9个高同源区段SNP所在的长片段,使用纯化后的第一轮PCR产物作为模板进行扩增子建库测序,检测样本共得2 928个SNP位点信息,测序成功率高达98.885 6%。利用Hardy-Weinberg(HWE)法则计算试验研究的9个高同源区段SNP位点的基因频率(p值均大于0.05,符合HWE法则),并与NCBI(national center for biotechnology information)中千人基因组数据库中获取的基因频率相比对,发现二者单碱基基因频率一致(误差限<0.15)。研究表明,利用多重长PCR靶向捕获技术结合二代测序技术为高同源区段的SNP分型提供一个准确、快速、大样本检测方案。