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。     

过量DMSO显著降低低模板浓度PCR扩增的特异性

DMSO通常经验性地用于提高PCR扩增的效率,但是过量的DMSO可以显降低特异序列的扩增效率,尤其是导致非特异性扩增的现象却被忽视。在6％的DMSO存在时,开始出现非特异条带,同时特异扩增产物减少。本首次报道DMSO对PCR产物特异性的影响并确定了克服上述现象产生的途径,即通过增加模板－引物的比例消除非特异条带。而通常提高复性温度只能部分减少非特异产物,不能避免非特异扩增。本结果有助于提高常规PCR,尤其是分子遗传学分析中经常使用的随机扩增多态性DNA（random amplifeid polymorphic DNA,RAPD）检测的准确度。     

影响反转录过程多种因素的探讨

本文探讨了影响反转录反应的各种因素。温度的适当提高和模板的严格处理可以破坏RNA的二级结构,并提高引物的特异性结合;模板的纯度可以避免杂质对酶的特异性的影响;巢式引物可以在PCR过程中很有效地避免反转录过程中产生的非特异性产物的扩增,而 提高最终产物的特异性。     

用三引物法提高PCR的扩增效率及特异性

聚合酶链反应(PCR)现已广泛应用于分子生物学研究的各个领域。对于模板量较低的样品及单拷贝基因,通常通过增加扩增的次数或多次PCR提高扩增的效率,但这样往往出现非特异性反应。本文采用三引物双扩增法大大提高了PCR扩增的效率及特异性。 1 材料与方法 Melanoma细胞由本室保存,能分泌人组织型纤溶酶原激活剂(t-PA)。RT PCR所用引物与t-PA     

添加扩增内标的沙门氏菌PCR检测方法

通过构建人工扩增内标,建立可以有效指示沙门氏菌检测过程可能出现假阴性情况的PCR检测方法。本研究基于沙门氏菌invA基因设计特异性引物,复合法构建扩增内标,建立PCR检测体系。特异性引物LW,对33株沙门氏菌和6株非沙门氏菌标准株进行检测,结果显示,所有沙门氏菌均扩增出385 bp的目标片段,非沙门氏菌则只能扩增出484 bp的扩增内标片段,特异性良好。灵敏度实验表明,该检测体系的灵敏度可达6.35 fg/μL。人工污染实验表明,起始染菌量为3.2 CFU/25 mL时,仅需8h增菌培养便可检出。大量食品样品检测证明,该检测体系确实可以有效的避免PCR检测过程出现的假阴性,提高检测准确性。     

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。     

纳米金增强复杂体系中PCR扩增低拷贝基因的反应特异性初步研究

目的：利用纳米金颗粒提高复杂体系基因组低拷贝基因PCR扩增的反应特异性。方法：首先,模拟复杂基因组扩增模式体系,以接近单拷贝的λDNA为模板,在PCR过程中加入纳米金颗粒,设计优化实验,以便模拟建立复杂基因组低拷贝目的基因PCR扩增的模式体系。随后,扩增人类基因组的疾病相关的低拷贝基因模板（如人基因组肿瘤坏死因子基因外显子1的380bp）,以检验纳米金优化增强PCR反应特异性的实际效果。结果：在复杂体系基因组的低拷贝基因PCR扩增中,纳米金颗粒能够较好地增强其PCR反应的特异性。结论：初步表明基于纳米金的纳米粒子PCR方法可以对复杂的实际基因组体系低拷贝基因的PCR扩增起到优化作用,这对于PCR反应优化方法的改进、推广具有重要的参考价值。     

高保真酶特异性检测大鼠SNP基因型新方法

目的应用高保真酶（Pfu）和3’末端修饰引物在单管双向等位基因特异性扩增（SB-ASA）中区分SNP基因型,建立高保真酶特异性检测SNP基因型的新方法。方法选取近交系大鼠SNP位点,以RS8149053为例,设计两个外部引物和两个等位基因特异性引物,四引物3’末端进行硫代磷酸化修饰,应用高保真聚合酶（Pfu）进行特异性扩增,扩增结果测序验证其可靠性。结果在RS8149053 SNP位点（C/T）上,等位基因型CC扩增出179 bp目的片段,基因型TT扩增出597 bp目的片段,基因型不同则扩增出分子量不同的片段,目的条带测序结果与Rat Genome Database数据库基因型结果一致,高保真酶扩增结果稳定且特异性强。结论高保真酶等位基因特异性扩增技术能有效降低假阳性率,是一种快速、特异的SNP基因分型新方法。     

小金海棠MxYSL5启动子克隆技术的比较

分别利用3种基于PCR技术的染色体步移法克隆了小金海棠MxYLS5基因的启动子,并比较了这三个体系的扩增产物。3种体系都可以获得特异性扩增产物,但扩增条带的长度和数量均不同,其中利用Genome Walking Kit扩增出来的条带特异性较好长度最长,而且特异性引物和简并引物的设计及退火温度的设置对3种体系的扩增产物起着关键的作用。基于PCR技术的染色体步移法为小金海棠MxYLS5基因启动子的克隆提供了一个稳定可靠的技术手段。     

羊驼性别决定基因sry部分片段的克隆与序列分析

从成年羊驼血液提取基因组DNA,参照哺乳动物sry(sex-determining region on the Y chromosome)基因的同源保守区域设计特异性引物,用PCR技术成功扩增羊驼sry基因的部分片段,且全部实验雄性个体均成功扩增,而雌性个体则无任何特异性片段,说明所扩增基因片段具雄性特异性。对扩增序列所编码蛋白序列分析显示所扩增的片段编码的蛋白序列在哺乳动物sryHMG-box(high mobility group box)蛋白超家族的HMG-box区域,说明扩增的为sry基因片段。用所扩增片段与其他哺乳动物同源序列分析显示,由sry基因构建的系统进化树,与传统的动物分类关系相近,说明由sry基因的HMG-box构建系统树是分析物种亲缘关系的有效工具。     

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87^N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.     

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.     

核酸恒温扩增技术研究进展

核酸恒温扩增技术在生命科学研究及相关诸多领域已经得到了广泛应用。我们对核酸恒温扩增技术的最新进展作一简要综述,包括环介导恒温扩增、链替代扩增、依赖核酸序列的扩增、滚环扩增、切口酶核酸恒温扩增、依赖解旋酶的恒温扩增、转录依赖的扩增、杂交捕获法、转录介导的扩增等的原理、优缺点及应用。     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.     

Anchored multiplex amplification on a microelectronic chip array

We have developed a method for anchored amplification on a microchip array that allows amplification and detection of multiple targets in an open format. Electronic anchoring of sets of amplification primers in distinct areas on the microchip permitted primer-primer interactions to be reduced and distinct zones of amplification created, thereby increasing the efficiency of the multiplex amplification reactions. We found strand displacement amplification (SDA) to be ideal for use in our microelectronic chip system because of the isothermal nature of the assay, which provides a rapid amplification system readily compatible with simple instrumentation. Anchored SDA supported multiplex DNA or RNA amplification without decreases in amplification efficiency. This microelectronic chip-based amplification system allows multiplexed amplification and detection to be performed on the same platform, streamlining development of any nucleic acid-based assay.     

全基因组扩增策略在基因芯片分析SARS冠状病毒实验中的应用

针对SARS冠状病毒的分子生物学检测是控制SARS流行的关键环节。为评价全基因组扩增对SARS微量样本检测的影响 ,采用 6 mer随机引物反转录 ,用加接头的随机引物合成第二链 ,再以接头序列为引物扩增并掺入荧光标记 ,最后与带有 70 mer探针的基因芯片杂交。此非特异方法基本覆盖了样本中的全部DNA ,结果发现SARS冠状病毒全基因组的扩增效果对基因芯片杂交结果的均匀性有较大影响 ,PCR循环次数增多会导致扩增均匀性的降低。分析了不同的引物对全基因组扩增均匀性的影响 ,探讨了全基因组扩增策略的缺陷。     

PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.