四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基引入了一个人为错配碱基 ,使引物的错误延伸率显著降低 ,大大提高了SNP分析的准确性 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .     

三色荧光技术在SNP检测中的应用

利用三色荧光标记的A、C、T双脱氧核苷酸单碱基延伸的方法结合编码寡核苷酸芯片技术检测单核苷酸多态性 (SNP)的基因型。以beta地中海贫血样本基因 (HBB基因 )突变作为模型的研究结果显示该方法能同时对多位点的SNP进行检测。     

一种新的基于单碱基延伸的SNP芯片技术

单核苷酸多态性(Single nucleotide polymorphism, SNP)是人类基因组中最常见的一种变异, 与疾病易感性、药物代谢等有着密切的关系。已经建立了多种SNP检测技术并得到了应用。单碱基延伸(Single base extension, SBE)是常用的SNP分型技术之一。文章建立了SBE结合Zip-code芯片技术对SNP进行分型, 为个体化用药及临床诊断芯片的研究与开发提供技术和方法。     

烟草种质基因分型核心SNP标记的开发

基于KASP(kompetitive allele specific PCR)技术平台,开发并验证可用于烟草核心种质基因分型的SNP(single nucleotide polymorphism)标记和对应检测引物,为烟草种质基因型鉴定评价、遗传多样性分析、核心种质筛选等提供技术和数据支持。利用Python和Perl脚本程序对覆盖烟草全基因组的1 179 154个SNP位点进行KASP引物设计和筛选,并通过试验验证其准确度和可用性。结果共有217 621个SNP位点完成了对应KASP引物设计,选择1 378个SNP位点进行试验验证,明确了732个可作为SNP标记,并确定48个SNP标记作为烟草种质资源基因分型的核心标记。这48个核心标记在烟草24条染色体上平均分布,平均PIC为0.36,平均MAF为0.39。利用确定的48个核心SNP标记,可以将各供试种质特别是将当前主栽烟草品种基因型进行区分,且标记具有极高的可靠性。     

PCR产物中无错误碱基拷贝最低比率的计算

分析了PCR过程中带有错误碱基拷贝的量变过程,得出不同循环(n)后不同类型拷贝数的计算通式并以逐次代入方式归纳出PCR产物中无错误碱基拷贝最低比率(R)和有效循环数(N),拷贝酶促合成链长(H)及错配率(f)的关系式R_n=(1-Hf/2)^N-1(1-Hf),对PCR技术制备表达用DNA片段有指导意义.     

荧光定量PCR诊断结核性脑膜炎的临床价值

目的 探讨荧光定量PCR检测脑脊液（CSF）结核分枝杆菌评估其临床意义。方法 应用荧光定量PCR技术检测45例CSF结核分枝杆菌并与抗酸杆菌染色。结核抗体酶联免疫吸附试验（结核抗体ELISA,简称酶联OT）进行比较。结果 荧光定量检测CSF结核杆菌的阳性率为11．1％,酶联OT为13．3％,抗酸杆菌染色为0％。结论 荧光定量PCR用于检测CSF结核分枝杆菌不具有灵敏度,阳性率低,对临床诊断结核性脑膜炎（简称结脑）无多少价值,不值得在临床上推广应用,必须寻找更有价值的诊断方法。     

实时荧光定量PCR的研究进展及应用

实时定量PCR(real-time PCR)的应用范围非常广泛,包括mRNA表达的研究、DNA拷贝数的检测、单核苷酸多态性(SNPs)的测定等。由于传统的PCR技术不能准确定量,使其在实际应用方面受到很大限制,因此,对PCR产物进行准确定量,尤其是病毒性病原的动态监控,成为迫切需要。     

实时定量PCR发展概述

实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,简称qPCR)是一种通过荧光信号对PCR进程进行实时监测,并对未知模板进行定量分析的一种核酸定量技术,该技术在临床诊断和生命科学等多领域发挥着重要的作用。现就生物制品领域有着重要应用价值的中介探针聚合酶链反应(mediator probe polymerase chain reaction,MP PCR)和数字聚合酶链反应(digital polymerase chain reaction,dPCR)新技术加以介绍,同时也对qPCR技术中的关键因素(如参考基因选择和核酸质量评价)以及qPCR最低限度标准(minimum information for the publication of real-time quantitative PCR,MIQE)指南作一概述。     

荧光定量PCR检测性病病原体的临床价值

目的荧光定量PCR检测性病病原体的临床意义.方法用荧光定量PCR(FQ-PCR)方法,对淋球菌(NGH)、沙眼衣原体(CT)、解脲支原体(Uu)3种性病病原体进行定量测定,其结果与定性PCR相比较.结果2种方法对3种病原体检测结果的符合率分别为NGH95.7%,CT96.9%,Uu95.2%,阳性率差异分别为4.3%,3.1%4.8%.两种方法阳性率差异无显著性(P＞0.05),阳性标本定量平均拷贝数分别为NGH 4.9×106,CT3.9×106,Uu 4.7×106.结论FQ-PCR操作更简便、快速,并且能准确定量,对于性病的诊断,病性的发展和愈后监测,疗效评价,指导临床用药具有很高的临床实用价值.     

等位基因特异PCR技术的研究与应用

生物的单核苷酸多态性(Single-nucleotide polymorphism,SNP)具有数量多、分布广、易于分型、稳定性强等优点,很适合于用做分子标记.等位基因特异PCR(Allele-specific PCR,AS-PCR)是根据SNP位点设计3'末端与SNP位点碱基互补或错配的特异PCR引物,通过凝胶电泳等方法检测PCR扩增产物的有或无,从而检测基因型中SNP的一种技术.经过不断地改进与完善,基于SNP的等位基因特异PCR标记已逐渐成为一种快速、简便、低成本、可靠、高通量的检测基因型SNP的方法.本文应用等位基因特异PCR技术,根据小麦TaDREB1基因在旱选10和鲁麦14的120(C→A)SNP成功地开发了一个SNP分子标记,证明了该方法的有效性和可行性.     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

等位基因特异性引物PCR技术及其应用研究

目的：研究建立等位基因特异性引物PCR技术体系,并将其应用于基因单核苷酸多态性研究工作。方法：通过美国国家生物信息中心(NCBI)的genBank获取基因序列及其相应位点的SNP信息。利用Primer5．0软件设计引物,并经NCBI的Blast2．0软件检验其特异性。结果：建立了单一等位基因特异性引物PCR(SASP—PCR)与嵌套式等位基因特异性引物PCR(NASP-PCR)两种技术,并应用于β2肾上腺素受体及内皮源性一氧化氮合酶基因单核苷酸多态性的研究,证实该技术的稳定性和优越性。结论：等位基因特异性引物PCR技术是一种更为简便、特异性较高、费用少的、便于推广的SNP检测方法,特别是在群体基因单核苷酸多态性研究中更有优势。     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.     

Heterogeneity of HLA-G Genes Identified by Polymerase Chain Reaction/Single Strand Conformational Polymorphism (PCR/SSCP)

A genomic HLA-G clone named 7.0E was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of α1 and α2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3′ untranslated region of 7.0E compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.0E-type were more heterogeneous than those of the G6.0- type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes. Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.     

纳米金对PCR扩增效率影响的机制探讨

近年来,纳米金粒子对聚合酶链式反应（polymerasechainreaction,PCR）的增效作用倍受关注,但是其具体的机制仍未明确提出。研究发现在PCR反应中,纳米粒子的增效作用是存在最佳浓度的,增加DNA聚合酶或者小牛血清蛋白（BSA）可以消除纳米金粒子导致的抑制。我们认为,纳米金粒子可能起到了类似于聚合酶B亚基的作用,提高了DNA聚合酶的延伸能力;而过量纳米金对PCR的抑制作用可能与纳米金结合单链DNA产生的位阻效应有关。     

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R~2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.     

一种快速构建cRNA标准曲线检测基因表达方法的建立

为了建立一种适于实验室乃至常规定量检测mRNA表达的、可快速构建cRNA标准曲线的方法,设计带有T7启动子序列和PolyT序列的引物对目的基因和内参照进行PCR,克隆入载体作为体外合成cRNA的模板,快速构建cRNA标准.结果表明：该曲线的线性范围至少达6个数量级,相关系数为0.99.该法快速、简便,适用于所有靶基因.