Thermal factors influencing detection of Vibrio vulnificus using real-time PCR

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.     

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.     

Multivariate Optimization of Polymerase Chain Reaction for Microbial Community Analysis

A multivariate regression, partial least square (PLS) approach was used to optimize a polymerase chain reaction (PCR) method for mixed communities. This approach, in contrast to univariate ones, provided information on the relative influence of the different factors to be optimized, as well as the interactions between factors. Models that predicted the outcome of further optimization were constructed from the initial experiments and verified experimentally. The models constructed were able to predict the outcome of a second set of experiments with high accuracy. PCR-amplification of DNA from environmental samples is often the first step in microbial community fingerprinting. Inhibitors and low cell numbers in the samples can cause problems with yield, for which compensation is normally made by increasing the number of cycles in the PCR-amplification reaction. Increasing the number of cycles, however, can cause other problems such as heteroduplex formation and increased bias. To avoid these problems the effects of different times of denaturing, annealing, and extension on yield were investigated for 2 different samples, one that consisted of a mixture of 9 laboratory strains, and one that represented the microbial community from the surface of the red alga Delisea pulchra. The multivariate approach showed, in addition to the successful optimization of yield, that the different factors affected the PCR depending on sample type. Annealing time had the largest effect on yield for the mixture of laboratory strains, whereas extension time was most important for the D. pulchra community. We suggest that multivariate optimization is a useful tool for PCR optimization and can be used irrespectively of the particular factors that are being investigated.     

Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA

PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/μL) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 μM each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 μM primers for Cyt b and COI, 0.3 μM primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 ° C for Cyt b, 56.1 ° C for 12S and 51.3 ° C for COI. PCR products obtained under these conditions produced excellent DNA sequences.     

PCR-amplification of GC-rich regions: 'slowdown PCR'

The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets. The protocol relies on the addition of 7-deaza-2'-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 degrees C s(-1) and a low cooling rate of 1.5 degrees C s(-1) for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures. The protocol takes 5 h to complete.     

Chemical composition, particle form and annealing temperature of amalgam alloy versus creep of the resulting amalgam

The influence of the chemical composition, particle form and annealing temperature of the amalgam alloy upon the creep of the resulting amalgam was investigated by means of a multiple regression model. Because no, or only little, information was available about the heat treatments of the different commercial alloys, the alloys were subjected to additional heat treatments at four different temperatures. The influence upon the creep of the amalgam of the Zn content, particle form and annealing temperature was found to be very significant. A higher Zn content as well as a spherical alloy resulted in less creep of the corresponding amalgam. The dependence of the creep upon the annealing temperature showed a minimum: more creep was obtained for amalgams prepared from the alloys heat treated at 150 degrees C and 435 degrees C than those from the alloys annealed at 315 degrees C and 319 degrees C, respectively.     

Micro Flow-Through Thermocycler with Simple Meandering Channel with Symmetric Temperature Zones for Disposable PCR-Devices in Microscope Slide Format

Chip-based flow-through PCR implements the PCR as a continuous process for nucleic acid analytics. The sample is transported in a winding channel through temperature zones required for denaturation, annealing and extension. Main fields of application are the monitoring of continuous processes for rapid identification of contaminants and quality control as well as high throughput screening of cells or microorganisms. A modular arrangement with five heating zones for flow-through PCR is discussed and evaluated. The special heater arrangement allows the implementation of up to 40 cycles on the footprint of a microscope slide, which is placed on top ofa 5 zones heating plate. Liquid/liquid two phase flow of PCR reaction mixture and mineral oil have been applied to create a segmented flow process scheme. In that way, the developed system may provide flow-through PCR as a unit operation for the droplet based microfluidics platform. The single use of disposable devices is commonly preferred due to the sensitivity of the PCR process to contaminations. All-glass microfluidic chips and disposable chip devices, made from polycarbonate as a replication with identically geometry, have been fabricated and tested. For the first time, microchannel geometries with nearly circular profile developed by all-glass technology have been transferred to mass fabrication by injection compression molding. Both devices have been successfully applied for the detection of the tumor suppressor gene p53. Although product yield and selectivity of the amplification process do not depend on the chip material, a well defined, reliable segmented flow regime could only be realized in the all-glass chip.     

Development of a simplified polymerase chain reactionenzyme immunoassay for the detection of Chlamydia pneumoniae

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae -specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.     

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.     

滨海电厂冷却水余热和余氯对中华哲水蚤的影响

依据滨海电厂冷却系统的实际运作情况,模拟研究了滨海电厂冷却水余热和余氯对中华哲水蚤的影响.结果表明：中华哲水蚤的热忍受能力随暴露时间延长而降低,随驯化温度升高而升高.驯化温度为16 ℃～27 ℃的中华哲水蚤在持续受到15、30、45 min热冲击及持续升温暴露24和48 h的致死温度分别为29.9 ℃～31.7 ℃、29.4 ℃～31.0 ℃、28.9 ℃～30.3 ℃和26.9 ℃～28.5 ℃、26.4 ℃～28.0 ℃.当驯化温度升高到一定程度后,其热忍受能力不再上升.电厂冷却水中余氯对中华哲水蚤的毒性随驯化温度升高、升温幅度增大及暴露时间延长而增强.     

Plastic versus glass capillaries for rapid-cycle PCR

Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area-to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees -7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225-300 bp in length with melting temperatures of 90.3 degrees -93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.     

Rapid cycle DNA amplification: time and temperature optimization

Rapid temperature cycling with hot air allows rigorous optimization of the times and temperatures required for each stage of the polymerase chain reaction. A thermal cycler based on recirculating hot air was used for rapid temperature control of 10-microliters samples in thin glass capillary tubes with the sample temperature monitored by a miniature thermocouple probe. The temperatures and times of denaturation, annealing and elongation were individually optimized for the amplification of a 536-base pair beta-globin fragment from human genomic DNA. Optimal denaturation at 92 degrees-94 degrees C occurred in less than one second; yield decreased with denaturation times greater than 30 seconds. Annealing for one second or less at 54 degrees-56 degrees C gave the best product specificity and yield. Non-specific amplification was minimized with a rapid denaturation to annealing temperature transition (9 seconds) as compared to a longer transition (25 seconds). An elongation temperature of 75 degrees-79 degrees C gave the greatest yield and increased yields were obtained with longer elongation times. Product specificity was improved with rapid air cycling when compared to slower conventional heat block cycling. Rapid thermal control of the temperature-dependent reactions in DNA amplification can improve product specificity significantly while decreasing the required amplification time by an order of magnitude.     

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.     

Neuro-genetic optimization of temperature control for a continuous flow polymerase chain reaction microdevice

In this study, a hybridized neuro-genetic optimization methodology realized by embedding finite element analysis (FEA) trained artificial neural networks (ANN) into genetic algorithms (GA), is used to optimize temperature control in a ceramic based continuous flow polymerase chain reaction (CPCR) device. The CPCR device requires three thermally isolated reaction zones of 94 degrees C, 65 degrees C, and 72 degrees C for the denaturing, annealing, and extension processes, respectively, to complete a cycle of polymerase chain reaction. The most important aspect of temperature control in the CPCR is to maintain temperature distribution at each reaction zone with a precision of +/-1 degree C or better, irrespective of changing ambient conditions. Results obtained from the FEA simulation shows good comparison with published experimental work for the temperature control in each reaction zone of the microfluidic channels. The simulation data are then used to train the ANN to predict the temperature distribution of the microfluidic channel for various heater input power and fluid flow rate. Once trained, the ANN analysis is able to predict the temperature distribution in the microchannel in less than 20 min, whereas the FEA simulation takes approximately 7 h to do so. The final optimization of temperature control in the CPCR device is achieved by embedding the trained ANN results as a fitness function into GA. Finally, the GA optimized results are used to build a new FEA model for numerical simulation analysis. The simulation results for the neuro-genetic optimized CPCR model and the initial CPCR model are then compared. The neuro-genetic optimized model shows a significant improvement from the initial model, establishing the optimization method's superiority.     

温度和盐度对蒙古裸腹Sou种群内禀增长能力的影响

报道了蒙古裸腹Sou(Moina mongolica)在20℃-33℃温度和5－40ppt盐度条件下和种群内禀增长率（rm),结果表明,20℃-30℃范围内蒙古裸腹Sourm随温度升高,超过30℃后继续升高,rm显著降低,在低盐度下蒙古裸腹Sou的种群增长能力相对较强,盐度为10ppt时rm最高,20－40ppt范围内Sou的rm差别不明显,本实验表明,25℃-30℃和10ppt分别是蒙古裸腹Sou种群增长较快的温度和盐度条件,在海水中长期培养对蒙古裸腹Sou的种群增长能力不会产生明显的不良影响。     

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。     

High-throughput PCR in silicon based microchamber array

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 °C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).     

Monitoring neurotensin[8-13] degradation in human and rat serum utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We have developed a novel method for rapid and empirical mapping of the protein interaction domain using a unique and atypical PCR-based amplification and a conventional yeast two-hybrid system. The modified PCR, designated as PASA-PCR, enables preferential amplification of the shortest amplicon from a complex expression library. PASA-PCR consists of reiterative cycles of denaturation of template DNAs and extremely abbreviated annealing/extension of primers to prevent their complete extension in a single cycle, followed by conventional amplification cycles. In PASA-PCR, the shortest (ranging from 400 to 1000 bp) amplicon is amplified almost exclusively from templates of various amplicon sizes. In addition, the frequency of in vitro recombination can be increased using low cooling rates (<0.5 degrees C/s) between the denaturation and annealing/extension steps, which was helpful in generating precisely trimmed protein-coding regions. Identification of Spc19-binding region of Spc34, which is a component of yeast's spindle pole body, was achieved by a combination of the yeast two-hybrid system and PASA-PCR.     

兰属SRAP-PCR反应体系的建立与优化

为获得兰属清晰的SRAP标记图谱,对兰属SRAP-PCR反应体系进行了初步探讨,建立了扩增多态性高、重复性好、带型清晰的SRAP-PCR反应体系。最佳反应体系：在30 μL反应总体系中,Mg2+ 2.2 mmol/L、dNTPs 0.8 mmol/L、DNA模板150 ng、DNA聚合酶2.0 U,上、下游引物各1.5 μmol/L;扩增程序：在94 ℃预变性4 min,反应前5个循环在94 ℃变性1 min、35 ℃复性1 min、72 ℃延伸1 min的条件下运行,随后的30个循环复性温度提高至55 ℃,最后72 ℃延伸5 min．     

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.