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℃。     

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。     

齿裂菌属和皮下盘菌属ISSR-PCR最佳反应条件的研究

在利用ISSR技术分析齿裂菌属和皮下盘菌属遗传多样性的研究中,为获得条带清晰、重复性好的ISSR扩增结果,对影响ISSR-PCR的条件进行了筛选,确定了此类菌物ISSR-PCR反应的最适宜条件:在15μLPCR反应体系中,10倍Taq酶缓冲液1.5μL,DNA模板8ng/μL,MgCl22.5mmol/L,dNTP0.15mmol/L,引物浓度0.4μmol/L,Taq酶1.00U,ddH2O9.0μL。最佳退火温度因不同的引物而定,最佳循环次数为35次。     

Systematic analysis of intrinsic factors affecting differential display

Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperaturefor a 13-mer arbitrary primer fell within a broad range of 40 degrees C-50 degrees C. Elongation times over a range of 30-120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2-50 microM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1-2.0 microM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression.     

杂交油菜ISSR-PCR反应体系的建立和优化

利用正交试验设计的方法,对影响ISSR-PCR反应的Mg²⁺、dNTP、引物和Taq DNA聚合酶4个因素进行优化试验,以期建立其最佳反应条件。并在此基础上,对DNA模板浓度和ISSR PCR反应程序中的退火温度进行梯度筛选。结果表明：杂交油菜20 μL ISSR-PCR最佳反应体系包括1.50 mmol·L^-1 Mg²⁺、0.125 mmol·L^-1 dNTP、2.00 μmol·L^-1 primer、0.50 U Taq DNA聚合酶、2.5 μL 10×buffer和40 ng DNA模板;引物UBC891适宜的退火温度为54.2℃。该体系在青杂3号及其父本不同个体中能够扩增出条带清晰、稳定性好的条带。ISSR-PCR反应体系的建立为利用分子标记技术研究杂交油菜品种纯度和真实性鉴定奠定了良好基础。     

黑木耳ISSR-PCR反应体系的正交优化

采用正交试验设计的方法,对黑木耳ISSR-PCR(简单重复序列区间-多聚酶链反应)反应体系中的5种主要因素(Taq聚合酶,Mg2+,模板DNA,dNTP及引物)4个水平进行优化筛选,确立了适合黑木耳ISSR分析的优化反应体系(20μL),通过梯度PCR试验筛选得到相应引物的最佳退火温度。     

Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures

Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products using thermostable polymerase produces fragments that are shorter than expected when the enzyme prematurely detaches as it approaches the 5′-end of the DNA template. These premature terminations result in a substantially reduced reading length of the DNA sequence. Since some DNA templates spontaneously fold and form stable secondary structures at temperatures that are typically used for primer annealing, one factor that may cause premature terminations to occur is the formation of secondary structures in the template during the annealing step of the cycle sequencing reaction. We describe a simple and effective method for reducing premature terminations in DNA sequences. We demonstrate that maintaining the annealing temperature of the cycle sequencing reaction above a critical temperature reduces premature terminations in DNA sequences that regularly contain premature terminations when the temperature of the annealing step is 60°C. In the method described, annealing and extension of the primer along the template take place at the same temperature (72°C). This procedure for reducing premature terminations can be applied when sequencing with primers that are relatively long (at least 27 mer) and have high optimal annealing temperatures.     

番茄叶霉病菌ISSR-PCR体系的建立

以番茄叶霉病菌(Passalora fulva)基因组DNA为模板,采用单因素试验和正交设计试验对该菌ISSR-PCR体系中的一些重要参数(Mg2+、dNTPs、引物、模板DNA、TaqDNA聚合酶、缓冲液、循环次数)和引物进行筛选和优化,并对退火温度进行了梯度优化,建立了番茄叶霉病菌ISSR-PCR的最佳反应体系(20μL):Mg2+1.5 mmol/L,dNTPs 0.4 mmol/L,引物1.5μmol/L,模板DNA45 ng,TaqDNA聚合酶1.0 U,1倍的PCR缓冲液,循环40次,退火温度50℃。     

荔枝DNA提取及RAPD扩增条件优化

为应用RAPD技术开展对荔枝种质资源的分析,以S43(GTCGCCGTCA)为引物,通过试验设计,分别研究了退火温度、模板浓度、引物浓度、dNTP浓度、Taq DNA聚合酶用量对荔枝RAPD-PCR反应的影响。建立并优化了适宜荔枝RAPD分析的扩增体系:20μL的反应体系,30ng的模板DNA度,0.25μmol/LRAPD引物、1.0UTaqDNA聚合酶,0.2μmol/LdNTP为荔枝适宜的RAPD-PCR扩增条件。     

Optimization of the conditions for RAPD-PCR of Candida spp. and Cryptococcus spp

In this study, we determined the optimal RAPD amplification conditions to obtain genetic molecular markers for the rapid and accurate identification of Cryptococcus spp. and Candida spp. The following parameters are modified: template DNA, DNA polymerase, magnesium cloride and primer concentration; denaturation, annealing and extension time, temperature of annealing and thermal cycles. After the optimization, reliable and reproducible RAPD patterns are obtained.     

黄连ISSR反应条件优化的研究

以黄连(味连,Coptis Chinensis Franch.)基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分（Mg²⁺、dNTP、引物、模板、Taq DNA聚合酶）以及热循环参数（退火温度、循环数、变性时间、退火时间、延伸时间）对扩增结果的影响,并找出各自的最适条件,建立了适合黄连ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCR buffer、1.5mmol·L^-1 Mg²⁺、200μmol·L^-1 dNTP、0.3 μmol·L^-1引物、40 ng模板、1 U TaqDNA聚合酶。扩增程序为94℃预变性5 min,然后进行35个循环：94℃变性30 s,（据不同引物的退火温度）复性1 min,72℃延伸1.5 min,循环结束后72℃延伸7 min,-4℃保存。这一优化系统的建立为今后利用ISSR标记技术进行黄连鉴定及种质遗传多样性分析提供了一个标准化程序。     

高分辨率熔解曲线法检测CYP4A118590T＞C单核苷酸多态性

目的 建立CYP4A11 8590T＞C单核苷酸多态性（single nucleotide polymorphism,SNP）的高分辨率熔解曲线（high resolution melting,HRM）检测方法.方法先采用温度梯度PCR,确定适宜的退火温度;再利用正交试验,优化引物、DNA模板量和Mg2＋量,最终确定PCR反应体系和反应条件.通过对607例无血缘关系的受试者基因组DNA进行HRM分析,并随机选择50例产物测序.结果 引物最适退火温度为57.8 ℃;PCR最佳反应体系为20 μl,包括2×conc dNTP mix 10 μl,上下游引物（10 μmol/L）各0.5 μl,DNA溶液（30 ng/μl）1.0 μl,Mg2＋（2.5 mmol/L）1.5 μl和灭菌水6.5 μl.607例受试者中CYP4A11 8590TT、TC和CC基因型频率分别为54.7 %、37.6 %和7.7 %.结论该正交试验优化的HRM技术可用于检测CYP4A11 8590T＞C单核苷酸多态性,且其分析结果和测序结果一致.     

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.     

Design of endonuclease restriction sites into primers for PCR cloning

I present a software system PCRCLNG that facilitates the design of endonuclease restriction sites into the 5'-end of PCR primers. The product amplified using these primers can be directly cloned into vectors. The program estimates the annealing temperature for each primer and selects the primer pairs with comparable annealing temperature. Finally the software determines whether the PCR product can be cloned into the vector to generate in-frame gene fusion.     

High-throughput sequencing of 16S rRNA gene amplicons: effects of extraction procedure, primer length and annealing temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.     

Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR method.

We describe a rapid and efficient megaprimer PCR procedure for site-directed mutagenesis that does not require any intermediate purification of DNA between the two rounds of PCR. This protocol is based on the design of forward and reverse flanking primers with significantly different melting temperatures ( T m). A megaprimer is synthesized in the first PCR reaction using a mutagenic primer, the low T m flanking primer and a low annealing temperature. The second PCR reaction is performed in the same tube as the first PCR and utilizes the high T m flanking primer, the megaprimer product of the first PCR and a high annealing temperature, which prevents priming by the low T m primer from the first PCR reaction. We have used this protocol with two different plasmids to produce cDNAs encoding seven distinct mutated proteins. We have observed an average mutagenesis efficiency of 82% in these experiments.     

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.     

濒危植物峨眉野连ISSR反应体系的建立与优化

针对峨眉野连ISSR的反应特点,建立稳定可靠的ISSR-PCR分子标记反应体系,为进一步研究峨眉野连的种质资源遗传多样性奠定基础。通过筛选引物并设定影响峨眉野连ISSR-PCR反应诸因子的不同梯度,检测其不同反应体系的扩增效果,分析非特异性条带的产生原因并进行条件优化,建立峨眉野连ISSR-PCR稳定可靠的反应体系。首次建立了可用于峨眉野连ISSR-PCR分析的最适宜的反应体系:25μLPCR反应体系中,内含1×PCRBuffer,1.5mmol/LMg2+,200μmol/LdNTP,0.3μmol/L引物,80ng模板,1.0UTaqDNA聚合酶。扩增程序为94℃预变性5min,然后进行35个循环:94℃变性30s,(据不同引物的退火温度)复性60s,72℃延伸90s,循环结束后72℃延伸7min,4℃保存。所建立的峨眉野连ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点,可以较好地应用于峨眉野连的种质资源多样性及居群鉴别的研究。     

小菜蛾中肠氨肽酶N3基因片断克隆技巧及其表达量

本研究通过设计简并引物,并且进行温度梯度PCR和二次PCR,最终筛选出适合扩增小菜蛾Plutella xylostella(L.)中肠APN3(氨肽酶N3,Aminopeptidase N3)片段的引物和温度,这为研究APN3的基因功能、APN3同APN其他同工酶之间的亲缘关系奠定了基础。通过比较电泳图谱,发现APN3在小菜蛾中肠中的表达量远远低于APN1、APN2和APN5的表达量。小菜蛾中肠APN同工酶氨基酸序列分析发现,APN3同APN1的亲缘关系最近,同APN2的亲缘关系最远。以上结果对于揭示Bt毒素作用于小菜蛾的机理以及小菜蛾对Bt毒素的抗性分子机制具有重要理论意义。     

Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

The quantification of the biological diversity in environmental samples using high‐throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator‐specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high‐throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.