重组PCR技术的应用及优化

重组PCR是通过DNA重叠序列的衔接作用,使多个DNA分子融合在一起的体外扩增技术。它使基因全序列的拼接、基因融合、基因破坏及启动子交换等DNA操作变得简单易行。如今重组PCR已成为DNA分析的有效利器。本研究通过重组PCR在分子进化、基因敲除及基因敲入、启动子研究和转基因植物转化载体的构建等方面的实际应用,分析了该技术的影响因素,并针对引物设计、DNA碱基重叠长度、温度参数等重要反应条件提出了优化方案。     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。     

应用PCR技术定向引入DNA小片段的策略

描述一种应用PCR技术定向引入DNA小片段和特异酶切位点的方法。为了获得m2/loxp66EGFPloxp71基因片段。根据EGFP基因序列,设计一对特异引物,上、下游引物分别引入m2/loxp66、loxp71序列和Xhol、Mlu1酶切位点。以pEGFPN1质粒为模板,采用PCR扩增以合成DNA双链,插入到克隆载体pMD18T。对重组子测序结果表明,实现了DNA小片段和酶切位点的定向引入。     

PCR技术的新进展

PCR是用于在体外快速酶促扩增特定DNA片段的技术,本文对PCR技术的一些新方法进行了简要综述,包括易错PCR,DNA shuffilng,原位PCR,电子PCR,固相PCR和PCR芯片。     

蛋白质定向进化技术的研究进展

蛋白质定向进化技术是蛋白质分子改造的一个重要策略.重点介绍了易错PCR、DNA改组等对编码蛋白质的基因进行随机突变和重组的技术,以及构建突变体库和高通量筛选的方法,并探讨了定向进化技术在蛋白质工程中的应用及前景.     

单分子PCR技术及其应用

常规PCR技术大都以大量DNA分子(一般为105个以上)为模板进行DNA扩增。新近发展起来的单分子PCR技术是一种以极少量DNA分子(1、5或10个分子)为模板进行扩增的技术。在高保真度DNA聚合酶作用下,单分子PCR技术能扩增出高度一致的DNA;在低保真度DNA聚合酶作用下,单分子PCR技术又能构建出含有大量突变基因的DNA库。该将介绍单分子PCR的基本原理、实验步骤、特点及其应用。     

超嗜热古菌Thermococcus eurythermalis A501编码B家族DNA聚合酶的生化特性及PCR应用

DNA聚合酶广泛应用于PCR技术,在生命科学研究及相关领域发挥重要作用。但目前商业化DNA聚合酶仍不能完全满足科研需要,有必要寻求高性能DNA聚合酶。文中克隆表达了超嗜热古菌（Thermococcus eurythermalis）A501来源的B家族DNA聚合酶基因（NCBI数据库基因登录号为TEU＿RS04875）、表征该重组蛋白的生化特性、评价了其PCR应用。将删除intein蛋白序列的DNA聚合酶（Teu-PolB）进行体外重组表达,经亲和层析和离子交换层析纯化获得Teu-PolB蛋白;利用5′端带荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳鉴定Teu-PolB的生化特性;以噬菌体λDNA基因组为模板,探究Teu-PolB的PCR应用。结果显示,Teu-PolB具有DNA聚合酶活性和3′→5′核酸外切酶活性,该酶在98℃下的半衰期约为2 h,热稳定性高。使用Teu-PolB进行PCR扩增,最适PCR缓冲液为50 mmol/L Tris-HCl pH 8.0,2.5 mmol/L MgCl₂,60 mmol/L KCl,10 mmol/L （NH<...     

用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

基于数字PCR的单分子DNA定量技术研究进展

数字PCR是一项针对单分子目标DNA的绝对定量技术．该技术是将含有DNA模板的反应溶液分配到大量独立的反应室中并且发生扩增反应,通过统计反应室中的阳性信号来定量DNA的拷贝数．DNA样品在反应室中随机和独立分布是单分子成功扩增和准确定量DNA拷贝数的关键因素．本文综述了数字PCR的发展历史、数字PCR与实时荧光定量PCR的区别,以及数字PCR在临床诊断、转基因成分定量、单细胞基因表达、环境微生物检测和下一代测序等方面的最新进展,并展望了该技术的应用前景．     

DNA改组的最新动态及应用前景

DNA改组(DNA shuffling)是目前最方便、有效的一种分子水平的体外定向进化技术,该技术同倾向错误PCR (Error-prone PCR) 相结合,通过对单基因或相关基因家族的靶序列进行多轮随机诱变、重组和高通量的筛选,可以有效富集正突变,去除负突变,提高突变文库的丰度,创造新基因和获得期望功能的蛋白质。DNA改组技术已在新药物等领域取得了广泛的应用,极大地推动了现代生物科学和生物技术的发展。该技术同计算机强大的数据分析系统相结合,将会为后基因组学的发展提供强有力的技术平台。     

PCR扩增试验的动力学数学模型

PCR技术已日趋成熟,但因为影响因素较多、反应过程比较复杂,直到目前PCR技术已创立近二十年,尚未能给出较好的描述PCR 反应的数学方法。我们根据它的基本原理提出了能够描述其反应过程的动力学方程：Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactiv×(n-n1)-Ntarg× (1+n×P)］×Cu×M,准确地描述了PCR反应的产物积累规律,建立了PCR反应的动力学数学模型。用动力学数学模型预测的PE 7700仪器的CT值与仪器的实际数值一致。动力学数学模型配合适当的监测设备可以构成自动化的PCR 定量仪器。PE 7700 仪器使用本动力学模型处理、分析数据,定量结果的准确性会更好。各实验室可根据各自的实验条件,由模型估算PCR产物数量,为PCR后产物继续处理提供较准确的数量信息。本模型阐明了PCR反应在多次循环后必然由指数扩增转变为线性扩增的分子基础,为定量PCR 提供了准确的计算方法。 Abstract:The PCR technique has been set up for nearly twenty years and is becoming more and more ripe.But because of the multiple influencing factors and complicated reaction procedures,no mathematical method that can describe the PCR reaction has been given.On the basis of its elementary principle,we suggested a kinetic equation to describe the reaction procedure,Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactive×(n-nl)-Ntarg×(1+n×P)］×Cu×M.This equation can describe correctly the accumulation rule of PCR product and thus build up the kinetic-mathematical model of PCR reaction.The predicted CT value of PE 7700 by the kinetic-mathematical model was in accordance with the real value detected by the machine.This kinetic-mathematical model accompanied by proper detecting equipment and computer could make an automatic PCR instrument,which would produce much better result.A laboratory can predict the amount of PCR product by this model and provide accurate information for further handling of PCR product according to its own condition.In this model,the molecular basis that PCR reaction is doomed to change from exponential amplification to linear amplification had been clarified.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

免疫聚合酶链反应技术的建立及其对甲胎蛋白的检测

免疫ＰＣＲ是一种新的具高敏感性的抗原检测技术,它类似传统的ＥＬＩＳＡ法,若与抗体连接的酶用ＤＮＡ片段代替,则该ＤＮＡ片段可用于ＰＣＲ扩增。以链酶亲合素搭桥将生物素标记的抗体与ＤＮＡ相连建立了免疫ＰＣＲ技术,并将其用于检测甲胎蛋白（ＡＦＰ）,其敏感性比ＥＬＩＳＡ法高１０４。因此,免疫ＰＣＲ有可能作为一种具高敏感性的检测手段用于临床早期诊断     

筛选转基因动物的新方法——PCR及其产物测序法

用特异性引物对肌球蛋白轻链2启动子(myosin light chain-2,MLC₂)-糜酶融合基因的转基因新生鼠鼠尾DNA进行PCR筛选, 低熔点琼脂糖凝胶电泳回收阳性样品PCR所扩出的DNA条带,纯化后用同一对引物中的一个进行单引物PCR测序,与所转外源基因序列比较,进一步确定整合有外源基因的阳性鼠.PCR及PCR 产物测序法检测转基因动物具有操作方便,灵敏度高及特异性强等优点.     

Chemically modified primers for improved multiplex polymerase chain reaction

Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance.     

新定量PCR数据处理方法的理论探讨

日新月异的生命科学技术的发展及临床医学科学研究的需求,一般的PCR技术已远远不能满足工作的需要。PE公司在进行了大量的PCR动力学研究的基础上,发现了利用荧光标记探针在PCR循环过程中积累的荧光强度达到仪器捡出阈值时,系统的初始模板数量与循环次数之间有线性关系,据此建立了目前的PE 7700 、PE 5700仪器的定量PCR技术,开创了PCR技术的新局面。但是由于这一技术的误差较大,尚不能满足生命科学及临床医学科学研究的需求,因此需要继续研究新的定量PCR技术。PCR动力学数学模型是根据PCR 技术的原理提出的,能够准确描述PCR反应产物分子数量积累规律的动力学方程,给出了PCR产物数量或者荧光强度与初始模板数量及其他反应条件间的函数关系。利用这一关系,根据PCR反应已积累的产物数量,可以实现准确的定量PCR分析,得到初始模板数量达到定量PCR的目的。使用动力学数学模型做定量PCR分析,其结果的误差仅与使用的荧光强度数值的精确度相关。使用精确到6位数的荧光强度数据,模板数自100~1 000 000区间定量结果的准确性可达99%以上。本文根据模拟实验数据进行了初步的定量PCR分析,结果提示,目前的定量PCR仪器使用PCR动力学模型理论处理分析数据,定量分析的结果会比目前的CT值方法在准确性方面提高几十倍以上,可以满足各方面研究工作误差水平的需要。 Abstract:Today standard PCR can't satisfy the need of biotechnique development and clinical research any more.After numerous dynamic research,PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700.But the error of this technique is too great to satisfy the need of biotechnique development and clinical research.A better quantitative PCR technique is needed.The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system.This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions,and can reflect the accumulating rule of PCR product molecule accurately.Accurate quantitative PCR analysis can be made use this function relation.Accumulated PCR product quantity can be obtained from initial template number.Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used.For an example,when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1 000 000,the quantitative result accuracy will be more than 99%.The difference of result error is distinct using same condition,same instrument but different analysis method.Moreover,if the PCR quantitative analysis system is used to process data,it will get result 80 times of accuracy than using CT method.     

快速PCR研究进展

PCR是最常用的分子生物学技术之一,通过变性、退火和延伸的循环来完成核酸分子的大量扩增。快速PCR就是基于普通PCR的工作原理,在保证PCR反应特异性、灵敏性、保真度的前提下,在更短时间内完成对核酸分子的扩增。近年来已经开展了许多有关方面的研究工作。本文将以DNA聚合酶的改进、添加剂的选择、热循环仪改进为重点内容,综述快速PCR技术的研究进展。     

Large mitochondrial repeats multiplied during the polymerase chain reaction

The number of repeats in repetitive DNA like micro‐ and minisatellites is often determined by polymerase chain reaction (PCR). When we counted repeats in an array of mitochondrial repeats in the cattle tick (Boophilus microplus) we found that the number of repeats increased during PCR. Multiplication of the repeats was independent of the primers used to amplify the region, the PCR annealing temperature and the length of the PCR product. The use of PCR to determine the number of repeats in arrays needs to be reassessed. For long repeats, a subset of samples should always be analysed by Southern blot hybridization to confirm the PCR results.