葡萄ISSR-PCR反应体系的建立与优化

采用正交设计L9(34)对影响葡萄ISSR-PCR反应体系的4个因素(dNTP、TaqDNA聚合酶、引物、模板DNA)在3个浓度水平上进行试验,并通过直观分析初步确定其反应体系;在此基础上,通过单因素试验探讨了dNTP、TaqDNA聚合酶、引物、模板DNA、退火温度及循环次数等因素或条件对葡萄ISSR-PCR扩增结果的影响,确定最佳反应水平。最终建立了葡萄ISSR-PCR扩增的最佳反应体系:在25μL的反应体系中,dNTP浓度0.2 mmol/L,TaqDNA聚合酶的用量0.5 U,引物浓度0.4mmol/L,DNA模板用量40 ng。反应程序:94℃预变性5 min;94℃变性1 min,52℃退火1 min,72℃延伸1 min 30 s,40次循环;最后72℃延伸10 min,10℃保存。     

怀地黄SRAP扩增体系的建立与引物的筛选

为建立适合怀地黄SRAP-PCR分子标记技术体系,通过单因子实验分别研究了DNA模板浓度、TaqDNA聚合酶浓度、Mg2+浓度、引物浓度以及dNTP浓度对怀地黄SRAP扩增反应的影响,确立了适合怀地黄SRAP最佳反应体系为:在25μL的反应体系中,模板DNA量20ng/25μL、2.5mmol/LMg2+、0.32μmol/L的上下游引物、0.30μmol/L的dNTP以及2.5UTaq酶,并利用确定的体系从88个引物组合中筛选出12对适合怀地黄SRAP-PCR反应的引物。     

多倍体罗汉果PCR-RFLP参数的优化与应用

以多倍体罗汉果DNA为材料,采用L16(4~5)正交组合试验和单因素梯度试验,研究Mg~(2+)、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25μL扩增反应体系中,10×Buffer 2.5μL,MgCl_2 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng;退火温度为56℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0μL,内切酶5.0U,PCR产物15μL,超纯水补至20μL;酶切时间2 h。     

丹参ISSR-PCR反应体系的建立与正交优化

利用正交试验设计的方法,从引物浓度、Taq DNA聚合酶浓度、Mg2+浓度、dNTP浓度4种因素3个水平,对丹参ISSR-PCR反应体系进行优化分析,并在此基础上对模板DNA浓度、PCR反应过程中的退火温度进行梯度检测。结果表明:20μL ISSR-PCR反应体系中各因素的最佳浓度为1×PCR buffer、200μmol/L dNTP、1.0μmol/L引物、1.5mmol/L Mg2+和1 U Taq DNA聚合酶,最佳模板DNA浓度为20～60ng,引物UBC 835的最佳退火温度为51.7℃。     

Improved PCR Performance Using Template DNA from Formalin-Fixed and Paraffin-Embedded Tissues by Overcoming PCR Inhibition

Formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.     

Sequencing PCR DNA amplified directly from a bacterial colony

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.     

曼地亚红豆杉RAPD反应体系与程序的优化

以曼地亚红豆杉为研究对象,采用L16(45)正交组合实验和单因素梯度实验对MgCl2、dNTP、随机引物、Taq酶、模板DNA浓度和退火温度、循环次数等影响RAPD扩增的重要因素进行优化,以期建立最优的RAPD反应体系与程序。实验结果表明,各因素最适条件为:25μLPCR反应体系中10×Buffer2.5μL,MgCl21.5mmol/L,dNTP0.2mmol/L,随机引物0.6μmol/L,Taq酶1.0U,模板DNA80ng;退火温度为37℃,循环次数为45次。     

正交设计优化东亚砂藓DDRT-PCR反应体系

利用正交实验设计L25（5^6）对东亚砂藓（Racomitrium japonicum）DDRT—PCR反应体系的6因素（Mg^2＋、dNTP、锚定引物、随机引物、模板DNA、Taq酶）在5个水平上进行优化实验。结果筛选出各反应因素的最佳体系（20μL）为：Mg^2＋2．25mmol／L、dNTP0．4mmol／L、锚定引物1．0μmol／L、随机引物0．7μmol／L、模板DNA1．6μL、Taq酶2．5U。对东亚砂藓DDRT—PCR最佳反应体系进行梯度PCR引物退火温度筛选,得到的最佳退火温度为45．4℃。该优化体系的建立,为进一步进行东亚砂藓抗旱基因的筛选与克隆等一系列分子研究提供了重要参考依据。     

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

唐古特大黄ISSR-PCR反应条件的优化

利用单因素试验对影响唐古特大黄ISSR-PCR扩增的重要参数进行优化,以期建立其最佳反应条件。结果如下:20μL反应体系包括1.5×PCR buffer(15mmol/LTris-HCl,75mmol/LKCl),1.00mmol/LMgCl2,0.6UTaq DNA聚合酶,0.125mmol/LdNTP,0.5μmol/L引物和30ng模板DNA;引物UBC888适宜的退火温度为57.4℃。ISSR反应条件的建立为利用分子标记技术研究唐古特大黄居群遗传多样性奠定了良好基础。     

Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases.

The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.     

罗汉果SRAP反应体系的建立与优化

建立适合罗汉果的SRAP-PCR扩增体系,为罗汉果的遗传图谱构建及基因定位奠定基础。实验对罗汉果SRAP-PCR反应体系的影响因素(引物,dNTP,Taq酶,Mg~(2+),模板DNA)在多个水平上进行优化试验,筛选出各反应因素的最佳水平,建立了罗汉果SRAP-PCR反应的最佳体系(10μL):引物0.6μmol/L、dNTP0.25 mmol/L、Taq DNA聚合酶0.5U、Mg~(2+)2.0 mmol/L和模板DNA 30 ng。该体系的建立能很好的满足罗汉果基因组DNA的扩增要求,SRAP标记应用于罗汉果遗传研究是可行的。     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

随机扩增多态DNA影响因素的研究

随机拉多态ＤＮＡ分析受诸多因素的影响,我们发现不同厂家制造的ＰＣＲ扩增仪,不同厂家出品的ＴａｑＤＮＡ聚合酶和ＰＣＲ缓冲液,ＲＡＰＤ反应体系中的引物浓度,Ｍｇ＾２＋浓度,ｄＮＴＰ浓度,ＢＳＡ和明胶,以及模板ＤＮＡ的量等均可能对ＲＡＰＤ结果有不同程度的影响。     

Sequence-Dependent Biophysical Modeling of DNA Amplification

A theoretical framework for prediction of the dynamic evolution of chemical species in DNA amplification reactions, for any specified sequence and operating conditions, is reported. Using the polymerase chain reaction (PCR) as an example, we developed a sequence- and temperature-dependent kinetic model for DNA amplification using first-principles biophysical modeling of DNA hybridization and polymerization. We compare this kinetic model with prior PCR models and discuss the features of our model that are essential for quantitative prediction of DNA amplification efficiency for arbitrary sequences and operating conditions. Using this model, the kinetics of PCR is analyzed. The ability of the model to distinguish between the dynamic evolution of distinct DNA sequences in DNA amplification reactions is demonstrated. The kinetic model is solved for a typical PCR temperature protocol to motivate the need for optimization of the dynamic operating conditions of DNA amplification reactions. It is shown that amplification efficiency is affected by dynamic processes that are not accurately represented in the simplified models of DNA amplification that form the basis of conventional temperature cycling protocols. Based on this analysis, a modified temperature protocol that improves PCR efficiency is suggested. Use of this sequence-dependent kinetic model in a control theoretic framework to determine the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is discussed.     

The enhancement of PCR amplification by low molecular-weight sulfones.

DNA amplification by polymerase chain reaction (PCR) is frequently complicated by the problems of low yield and specificity, especially when the GC content of the target sequence is high. A common approach to the optimization of such reactions is the addition of small quantities of certain organic chemicals, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol and formamide, to the reaction mixture. Even in the presence of such additives, however, the amplification of GC-rich templates is often ineffective. In this paper, we introduce a novel class of PCR-enhancing compounds, the low molecular-weight sulfones, that are effective in the optimization of high GC template amplification. We describe here the results of an extensive structure-activity investigation in which we studied the effects of a series of six different sulfones on PCR amplification. We identify two sulfones, sulfolane and methyl sulfone, that are especially potent enhancers of high GC template amplification, and show that these compounds often outperform DMSO and betaine, two of the most effective PCR enhancers currently used. We conclude with a brief discussion of the role that the sulfone functional group may play in such enhancement.