高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

PCR技术检测人乳头状瘤病毒DNA

本文用一对适合人乳头状瘤病毒DNA的通用PCR引物对临床阳性和正常组织进行了PCR扩增检测分析,阳性组织均得到一条特异性的DNA扩增片段,正常组织均没有任何扩增片段。阳性组织DNA扩增片段经克隆后进行DNA序列分析,证明该扩增片段确为目标DNA扩增片段。     

西部马脑炎病毒基因组序列的半套式RT-PCR检测

为进一步提高RT－PCR检测西部马脑炎病毒（WEE）病毒基因组方法的敏感性,采用半套式PCR扩增病毒基因组特异序列,首先采用逆转录法将病毒基因组RNA逆转录为cDNA,然后以此cDNA为模板,进行扩增。对扩增后电泳检查无可见DNA条带的产物进行半套式PCR;与此同时对扩增的循环数、Mg^ 浓度和退火温度等条件进行了优化,以进一步提高扩增的特异性。结果第一轮PCR未扩出特异笥片段的WEE病毒稀释度,其半套式扩增出特定大小的DNA产物;同时优化的条件提高了扩增产物的特异性。扩增产物约为190bp的单一DNA片段,其大小与预期的相一致,结果表明采用半套式RT－PCR方法检测WEE病毒的基因组序列的敏感性可提高100倍以上。     

利用PCR技术鉴定河豚鱼的探索

目的:利用聚合酶链反应(PCR)技术,建立鱼类制品中河豚鱼成分的分子生物学鉴定方法,弥补河豚鱼形态学鉴定的缺陷。方法:通过阅读文献,确定河豚鱼鉴定的靶基因并筛选特异性引物,提取河豚鱼DNA,优化PCR反应条件,扩增河豚鱼特异性DNA片段,并通过DNA电泳显示特异性扩增的DNA条带。结果:选择细胞色素氧化酶亚基Ⅰ(COⅠ)作为河豚鱼成分鉴定的靶基因,PCR最佳反应条件是退火温度68℃,镁离子浓度2.0 mmol/L,DNA模板加入量0.07～0.1μg。河豚鱼特异性引物具有较好的特异性,与市场上常见的鲤鱼、草鱼等水产品无交叉反应。结论:所建立的PCR方法可以有效鉴定鱼类制品中河豚鱼成分。     

PCR技术在检测鼠金黄色葡萄球菌中的应用研究

目的　建立实验大小鼠金黄色葡萄球菌的快速检测方法———PCR法。方法　根据已公布的金黄色葡萄球菌耐热核酸酶nuc基因的序列 ,设计并合成一对特异性的引物 ,利用PCR技术扩增nuc基因片段。对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株抽提的DNA进行扩增。结果　金黄色葡萄球菌PCR产物出现 6 6 8bp的特异性DNA扩增片段 ,而其他非金黄色葡萄球菌未出现扩增片段 ,证实了合成的引物对金黄色葡萄球菌具有特异性。将抽提的金黄色葡萄球菌DNA进行系列稀释 ,测定此PCR体系的敏感性 ,结果显示 ,该PCR体系能检出 3pg金黄色葡萄球菌DNA ,且从抽提DNA到PCR扩增及电泳结束仅需 4h。结论　本研究所建立的扩增耐热核酸酶nuc基因检测鼠金黄色葡萄球菌的PCR方法 ,具有快速、可靠、敏感和特异的特点 ,可用于临床样品和金黄色葡萄球菌感染时的检测 ,适合应用于实验大小鼠的监测。     

甜菜碱增强长片段PCR的扩增

聚合酶链式反应 (PCR)作为一项非常成熟的技术可以用于基因组序列的扩增。普通的PCR技术只适合于短片段DNA的扩增 ,一般在 6kb以下。对于 6kb至十几kb甚至几十kb以上的DNA片段的扩增就非常困难。通过添加不同化学物质 ,发现甜菜碱对长片段PCR的扩增有非常有效的增强作用。通过对玉米总DNA以及质粒DNA的扩增 ,发现 1mol L到 25mol L甜菜碱对改进PCR扩增效果明显。通过添加甜菜碱 ,可以从玉米基因组中扩增出 9kb以上的单拷贝片段 ,从质粒中扩增出 16kb以上片段。经过试验 ,发现不同GC含量的引物需要使用不同浓度的甜菜碱。甜菜碱可以减少甚至消除长片段PCR中的非特异性扩增。同时 ,我们发现其它的添加物 ,如DMSO ,甘油 ,甲酰胺对长片段PCR的作用不明显     

甜菜碱增强长片段PCR的扩增

聚合酶链式反应(PCR)作为一项非常成熟的技术可以用于基因组序列的扩增。普通的PCR技术只适合于短片段DNA的扩增,一般在6kb以下。对于6kb至十几kb甚至几十kb以上的DNA片段的扩增就非常困难。通过添加不同化学物质,发现甜菜碱对长片段PCR的扩增有非常有效的增强作用。通过对玉米总DNA以及质粒DNA的扩增,发现1mol／L到2．5mol／L甜菜碱对改进PCR扩增效果明显。通过添加甜菜碱,可以从玉米基因组中扩增出9kb以上的单拷贝片段,从质粒中扩增出16kb以上片段。经过试验,发现不同GC含量的引物需要使用不同浓度的甜菜碱。甜菜碱可以减少甚至消除长片段PCR中的非特异性扩增。同时,我们发现其它的添加物,如DMSO,甘油,甲酰胺对长片段PCR的作用不明显。     

一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。     

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。     

福尔马林固定白鱀豚标本DNA提取及其遗传多样性的初步研究

福尔马林固定标本是宝贵的遗传资源,但是如何有效利用其中的遗传信息一直存在问题。本文尝试从标本预处理、消化、PCR扩增各方面综合考虑和优化改进,成功提取并扩增21头福尔马林固定白豚标本线粒体DNA控制区410bp片段。采用了3种预处理方法尽量去除固定标本中残存的甲醛,从试验结果来看,从酒精梯度 临界点干燥处理的标本中提取的DNA在扩增时具有明显优势。通过蛋白酶K消化过程中对于酶的浓度、温浴时间的比较试验,发现随着采用大幅提高酶浓度、延长消化时间等高强度的蛋白酶消化操作后,DNA的质量和产量均得到显著提高。针对标本DNA降解严重的特点,设计特异性好且长度合适的引物以及使用巢式引物扩增,均提高了标本DNA扩增的特异性和灵敏度。通过对所测得的21头白鱀豚线粒体DNA控制区部分序列的对比,发现全部个体在该片段上的序列完全一致,说明白豚遗传多样性极低。     

Energy transfer cassettes for facile labeling of sequencing and PCR primers

Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the design, synthesis and evaluation of a four-color set of ET cassettes, fluorescent labeling reagents that can be quantitatively coupled to a thiol-activated target through a disulfide exchange reaction. The ET cassette consists of a sugar-phosphate spacer with a FAM donor at the 3'-end, an acceptor linked to a modified T-base at the 5'-end of the spacer and a mixed disulfide for coupling to a thiol at the 5'-end. The acceptor dye emission intensities of ET labeled primers produced in this manner are comparable to commercial ET primers. The utility of our ET cassette-labeled primers is demonstrated by performing four-color capillary electrophoresis sequencing with the M13(-21)forward primer and by generating and analyzing a set of single-nucleotide-polymorphism-specific PCR amplicons.     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

Red/ET重组及其在生物医学中的应用

通过应用Rac噬菌体的RecE RecT和λ噬菌体的RedαRedβ系统而建立的DNA工程平台———Red ET重组,是一种不依赖于限制性内切酶的分子克隆新技术。运用该技术能够介导PCR产物或寡核苷酸对目标基因进行剪切、插入、融合及突变等多种操作,在生物医学领域里具有广阔的应用前景,尤其在基因组功能研究中对BACs、PACs和细菌染色体的打靶修饰以及基因敲除动物DNA靶分子的快速构建等方面最有效。随着Red ET重组的推广与应用,该技术本身也在不断被改进,在工作效率得到显著提高的同时,其操作也变得更加简单、省时、省力。结合自身的一些研究结果,对Red ET重组的技术特点、发展现状和在生物医学中的应用进行了详细阐述。     

DNA cloning by homologous recombination in Escherichia coli

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.     

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.     

Factors affecting mitochondrial DNA quality from museum preserved Drosophila simulans

In this study we investigate how traditional killing methods and storage regimes affected mitochondrial DNA quality in Drosophila simulans. Here we define quality with three criteria: (1) size of extracted DNA, (2) extraction yield, and (3) ability to amplify from four target regions. Killing methods had a significant effect on extraction yield, but not on PCR success. Highest DNA yields were extracted from specimens exposed to cyanide, while the lowest were from specimens killed in 70% ethanol. Specimens stored for two years contained badly sheared DNA, which translated into a significant decrease in PCR success compared to freshly assayed specimens. The most dramatic decrease in PCR success occurred in the 1822 bp and 1332 bp amplicons, compared to the 959 bp and 291 bp fragments. Naphthalene did not affect any aspect of DNA quality; time of storage affected PCR success regardless of naphthalene environment. This study serves to further refine our understanding of DNA degradation.     

Evaluating the effects of laboratory protocols on eDNA detection probability for an endangered freshwater fish

The effectiveness and accuracy of detection using environmental DNA (eDNA) is dependent on understanding the influence laboratory methods such as DNA extraction and PCR strategies have on detection probability. Ideally choice of sampling and extraction method will maximize eDNA yield and detection probability. Determining the survey effort required to reach a satisfactory detection probability (via increased PCR replicates or more sampling) could compensate for a lower eDNA yield if the sampling and extraction method has other advantages for a study, species or system. I analysed the effect of three different sampling and extraction methods on eDNA yield, detection probability and PCR replication for detecting the endangered freshwater fish Macquaria australasica from water samples. The impact of eDNA concentration, PCR strategy, target amplicon size and two marker regions: 12S (a mitochondrial gene) and 18S (a nuclear gene) was also assessed. The choice of sampling and extraction method and PCR strategy, rather than amplicon size and marker region, had the biggest effect on detection probability and PCR replication. The PCR replication effort required to achieve a detection probability of 0.95, ranged from 2 to 6 PCR replicates depending on the laboratory method used. As all methods yielded eDNA from which M. australasica was detected using the three target amplicons, differences in eDNA yield and detection probability between the three methods could be mitigated by determining the appropriate PCR replication effort. Evaluating the effect sampling and extraction methods will have on the detection probability and determining the laboratory protocols and PCR replication required to maximize detection and minimize false positives and negatives is a useful first step for eDNA occupancy studies.     

Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.     

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.