非全长链合成导致连续聚合酶链式反应(PCR)扩增失败和复杂产物的形成

在进行连续PCR(以产物为模板进行多轮次的连续重复PCR扩增)实验时, 我们观察到了一种新的异常现象—— 用不同来源的模板连续PCR扩增不同长度的靶序列最终都会导致扩增失败, 表现为在常规琼脂糖电泳检测时特异产物条带的消失和不能泳动出点样孔之复杂异常产物的出现. 连续PCR扩增失败的时期依扩增靶序列的长度不同而不同, 越长的靶序列在连续PCR中扩增失败的时期越早. 扩增得到的复杂产物主要由连续分布的小于靶序列长度的具有相当程度多样性的非全长链组成. 复杂产物在内部具有局部的双链区域和大量的单链区域而在外部具有单链分支结构, 能够被单链特异的S1核酸酶消化, 但是不能被双链特异的限制性内切酶消化. 人工处理完整双链形成的非全长链长产物与完整双链以不同比例混合后作为模板进行连续PCR, 结果表明同源的非全长链成分对PCR扩增有严重的干扰作用. 已有的证据表明PCR扩增过程中形成的非全长链成分是导致这种异常现象的关键因素, 多个不同长度的非全长链复性形成“杂种分子”最终表现为复杂产物. 连续PCR扩增失败、PCR介导重组和长片段PCR难于操作有共同的产生基础—— 扩增过程中非全长链成分的产生. 任何降低PCR扩增过程中非全长链成分产生的措施, 特别是聚合酶忠实性的提高, 都能缓解异常扩增产物的出现和利于长片段PCR操作.     

优化重叠PCR法进行单链抗体基因扩增和点突变

利用重叠PCR技术扩增单链抗体基因或位点突变是抗体文库构建或稳定表达的关键和难点,国内外文献未见其方法学的系统报道.以不同VH、VL和Linker基因为拼接模板进行重叠PCR,针对影响重叠PCR扩增的拼接类型,引物设计,反应条件等进行优化.结果表明两段重叠连接比三段更容易实现,且扩增效果好;引物的互补序列长度一般应大于15 bp,且在18～24 bp 时扩增效果最好;退火温度在52～60℃,Mg2+浓度在1.5～2.5 mM时对拼接的效果影响较小;直接或间接使用拼接模板均可以实现重叠PCR的扩增.利用优化策略,首次构建了抗除虫菊酯的scFv基因文库并引入抗XAC糖蛋白scFv基因的点突变,为除虫菊酯抗体文库构建和抗XAC重组抗体的稳定表达奠定了基础.     

RT-SHIV rt基因单拷贝PCR扩增方法的建立及初步应用

目的建立RT-SHIV病毒全长rt基因单拷贝PCR扩增方法,用于HIV-1 rt基因体内遗传与变异研究。方法 Oligo软件设计RT-SHIV rt基因特异性扩增引物,梯度稀释方法进行特异性和灵敏度筛选,进而优化退火温度和PCR反应最佳循环数等条件,建立rt基因PCR扩增方法;在此基础上将模板进行有限稀释,摸索rt基因单拷贝PCR扩增条件;使用该方法扩增感染猴体内RT-SHIV病毒rt基因,BioEdit软件进行基因序列分析。结果筛选得到一组巢式PCR引物,成功建立了RT-SHIV rt基因PCR扩增方法;当模板浓度为100 copies/μL时,扩增产物为单拷贝序列;测序结果显示RT-SHIV感染猴d266和d294血浆样本分别存在1处和6处氨基酸突变。结论本研究建立的全长rt基因单拷贝PCR扩增方法特异性好、灵敏度高、重复性强,可以应用于各类RT-SHIV病毒的全长rt基因分析。     

一步法定点突变技术快速构建bsh基因突变启动子

目的：建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法：以构建单核细胞增生李斯特菌（Listeria monocytogenes）中编码胆碱水解酶（bile salt hydrolase,BSH）的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果：通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论：该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。     

采用单碱基突变模板作为内对照对组织中mRNA水平进行PCR定量

用PCR方法对原始模板进行单碱基突变,在原始模板DNA的特定位点引入一个EcoRI酶切位点。单碱基突变的DNA经PCR扩增后定量、稀释,作为内标加入到样品中与待测DNA同时进行PCR扩增．扩增产物经酶切后电泳,根据电泳结果中不同分子量DNA片段的含量,对样品中待测基因拷贝数进行定量分析。实验结果观察到：每1μg肝脏组织总RNA经逆转录后AVPV1受体cDNA拷贝数约1．25×10－20mol。     

PCR-mtDNA技术鉴别检测不同动物肌肉组织和饲料中鸭源性成分

以鸭肌肉组织DNA为模板,利用PCR-mtDNA技术成功克隆出了鸭mtDNA COIII基因(GenBank Accession No.DQ655706).对所克隆的序列分析表明.其序列包括鸭细胞色素C氧化酶III(COIII)基因全序列784 bp,通过同源性分析可知,动物的线粒体DNA COIII基因是相对保守的,利用此特性设计PCR-mtDNA方法鉴别检测鸭源性成分的特异性引物;以各种动物肌肉组织及饲料DNA为模板进行PCR扩增、经反复验证筛选出只能扩增出鸭DNA的目的片段,而不能扩增出其他动物DNA片段的特异性强、稳定性好的引物P3、P4;利用此引物PCR扩增鸭DNA的特异性片段为226 bp,对PCR产物进行测序分析可知与已克隆的鸭mtDNA COIII基因同源性达到100%,证明了所筛选引物的准确性.通过对不同含量的DNA模板溶液进行PCR扩增的方法,对筛选出的特异性引物P3、P4进行灵敏度试验,结果分析表明灵敏度约为0.001%,证明该PCR方法具有特异性强、灵敏度高的特点,完全可作为鉴别不同动物肌肉组织和饲料中鸭源性成分的方法.     

高温烹饪对动物肌肉组织DNA降解的影响

目的 探索高温烹饪对肌肉组织DNA降解程度的影响.方法 对牛肉、猪肉和鸡肉样本分别进行以下处理:不同温度烘烤0.5h;100℃沸水中加热不同时间;分别用水煮、油煎、红烧、烘烤烹饪肌肉至熟.分别提取DNA,PCR扩增线粒体DNA上的12S rRNA基因片段,电泳检测PCR产物的变化,并进行DNA序列测定.结果 样品经过不同温度处理均能扩增到目的条带,但条带从140℃开始显著减弱;沸水持续加热20 min、40 min后,3种肉的PCR产物量无显著影响,但加热80 min后,猪肉和鸡肉的PCR扩增产物条带明显减弱;红烧后的肌肉PCR扩增产物量显著减少.结论 温度、烹饪时间及烹饪方式对模板DNA的降解和后续PCR扩增有一定影响,但不影响肌肉组织的DNA可检测性.     

部分扩增与双片段连接相结合制作长基因定点突变

重叠延伸PCR是基因定点突变的主要方法,但是以该方法制作长基因定点突变时,往往遇到难以获得第二轮PCR产物或容易引入新的非预期突变等问题。此时,可先以重叠延伸PCR扩增含突变位点的部分基因片段,再将其连入适当载体获得重组质粒。若该扩增片段两侧的酶切位点在质粒载体上不单一,则可采用双片段连接法构建完整质粒。以制作视网膜母细胞瘤基因S780E定点突变为例,直接以重叠延伸PCR扩增全长基因时未能得到理想的目标产物。故先扩增含点突变的F3片段,再将其与源自原始质粒的F2片段一起连入含F1片段的质粒载体而构建完整质粒。两个筛选出的重组质粒经序列检测完全符合目标突变序列特征,验证了该方案的可行性。该方法作为重叠延伸PCR的补充,可为许多长基因定点突变提供解决方案。     

钩端螺旋体赖型017鞭毛钩相关蛋白K基因的克隆及序列分析

在以抑制消减杂交比较强毒株赖型钩端螺旋体017株和无毒株双曲钩体Patoc　I株　的基因组差异时,获得了一系列仅存在于强毒株而无毒株缺如的差异片段.选取差异片段AF325810设计特异性引物,以赖型钩端螺旋体017株基因组DNA为模板,进行巢式PCR扩增,PCR纯化产物T载体克隆,选取阳性克隆测序,进一步进行生物信息学分析,以获得强毒株赖型钩端螺旋体017株特有的毒力相关基因,DOT BLOT显示其在钩端螺旋体各株间有不同分布PCR扩增得到了产物为2kb大小的DNA片段,序列分析结果显示得到了问号钩端螺旋体赖型017株的鞭毛钩相关蛋白K基因的上游序列,为进一步探索钩端螺旋体的致病机制奠定了基础.     

植物叶绿体与线粒体基因组DNA特异性比较研究

研究不同植物间叶绿体和线粒体基因组DNA的差异,探讨其在法庭科学中的应用价值.根据叶绿体和线粒体基因组DNA核苷酸序列的特点,分别设计了一系列相应的引物,经PCR扩增后,电泳鉴别不同的植物.结果表明在设计的一系列引物中,叶绿体基因组DNA的PCR产物差异不大,鉴别效果不明显;而线粒体基因DNA的PCR产物差异大,鉴别效果明显.因此以线粒体DNA为模板进行PCR扩增,在不同植物间存在良好的差异性,适合于不同植物间的鉴别,在法庭科学中具有实际应用价值.     

A quantitative approach for polymerase chain reactions based on a hidden Markov model

Polymerase chain reaction (PCR) is a major DNA amplification technology from molecular biology. The quantitative analysis of PCR aims at determining the initial amount of the DNA molecules from the observation of typically several PCR amplifications curves. The mainstream observation scheme of the DNA amplification during PCR involves fluorescence intensity measurements. Under the classical assumption that the measured fluorescence intensity is proportional to the amount of present DNA molecules, and under the assumption that these measurements are corrupted by an additive Gaussian noise, we analyze a single amplification curve using a hidden Markov model(HMM). The unknown parameters of the HMM may be separated into two parts. On the one hand, the parameters from the amplification process are the initial number of the DNA molecules and the replication efficiency, which is the probability of one molecule to be duplicated. On the other hand, the parameters from the observational scheme are the scale parameter allowing to convert the fluorescence intensity into the number of DNA molecules and the mean and variance characterizing the Gaussian noise. We use the maximum likelihood estimation procedure to infer the unknown parameters of the model from the exponential phase of a single amplification curve, the main parameter of interest for quantitative PCR being the initial amount of the DNA molecules. An illustrative example is provided. This research was financed by the Swedish foundation for Strategic Research through the Gothenburg Mathematical Modelling Centre.     

The real-time polymerase chain reaction

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.     

Enhanced PCR amplification of VNTR locus D1S80 using peptide nucleic acid (PNA).

Use of the polymerase chain reaction (PCR) to amplify variable numbers of tandem repeat (VNTR) loci has become widely used in genetic typing. Unfortunately, preferential amplification of small allelic products relative to large allelic products may result in incorrect or ambiguous typing in a heterozygous sample. The mechanism for preferential amplification has not been elucidated. Recently, PNA oligomers (peptide nucleic acids) have been used to detect single base mutations through PCR clamping. PNA is a DNA mimic that exhibits several unique hybridization characteristics. In this report we present a new application of PNA which exploits its unique properties to provide enhanced amplification. Rather than clamping the PCR, PNA is used to block the template making it unavailable for interstrand and intrastrand interactions while allowing polymerase to displace the PNA molecules and extend the primer to completion. Preferential amplification is reduced and overall efficiency is enhanced.     

Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR‐based single‐molecule sequencing approach

While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.     

Solid phase DNA amplification: a Brownian dynamics study of crowding effects

Solid phase amplification (SPA), a new method to amplify DNA, is characterized by the use of surface-bound primers. This limits the amplification to two-dimensional surfaces and therefore allows the easy parallelization of DNA amplification in a single system. SPA leads to the formation of small but dense DNA brushes, called DNA colonies. For a molecule to successfully duplicate itself, it needs to bend so that its free end can find a matching primer, located on the surface. We used Brownian dynamics simulations (with a united-atom model) to model the basic kinetics of an SPA experiment. The simulations mimic the temperature cycles and the molecule duplication process found in SPA. Our results indicate that the steric interaction between molecules leads to a decreased duplication probability for molecules in the center of a colony and to an outward leaning for the molecules on the perimeter. These effects result in slower amplification (compared to solution PCR) and indicate that steric interaction alone can explain the loss of the exponential growth (characteristic of solution PCR) of the number of molecules in an SPA experiment. Furthermore, the growth of the colony as a function of the number of thermal cycles is found to be similar to the one obtained with a simple Monte Carlo simulation.     

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.     

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．     

Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains

DNA shuffling and other in vitro recombination strategies have proven highly effective at generating complex libraries for mutagenesis studies. While most recombination techniques employ DNA polymerases in part of a multi-step process, few seek to exploit the natural recombinogenic tendencies and exponential amplification rates of PCR. Here, we characterize a simple but effective method for using standard PCR to promote high recombination frequencies among compact heterologous domains by locating the domains near one end of the template. In a typical amplification reaction, Pfu polymerase generated chimeric crossover events in 13% of the population when markers were separated by only 70 nt. The fraction of recombinant sequences reached 42% after six consecutive rounds of PCR, a value close to the 50% expected from a fully shuffled population. When homology within the recombinant region was reduced to 82%, the recombination frequency dropped by nearly half for a single amplification reaction and crossover events were clustered toward one end of the domain. Surprisingly, recombination frequencies for template populations with high and low sequence homologies converged after just four rounds of PCR, suggesting that the exponential accumulation of chimeric molecules in the PCR mixture serves to promote recombination within heterologous domains.