Real-time PCR assay for measurement of mouse telomeres

Measurement of telomeres by polymerase chain reaction (PCR) amplification has been problematic due to the formation of dimers by the primers designed to hybridize to the telomere repeats. Recently, a set of primers that overcome this problem has been created and used to develop an assay to measure human telomeres by real-time quantitative PCR. We modified this assay to measure mouse telomeres. Results showed that the primers do indeed amplify mammalian telomere repeats without forming dimers. Results obtained from the real-time quantitative PCR assay of mouse DNA were similar to terminal restriction fragment analysis by pulsed-field gel electrophoresis followed by Southern hybridization. The assay performed with mouse DNA in a similar manner as it performs with human DNA. Preliminary linkage mapping suggests a gene influencing telomere length on the X chromosome. This assay will aid in the study of telomere function and importance in diseases associated with aging and cancer formation.     

Mycoplasma detection by PCR analysis

Summary The polymerase chain reaction (PCR) method was used to detect mycoplasma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chronically infected and 13 cell lines were negative. Sets of outer and inner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonucleotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium bromide-stained gels, both after the first round of amplification as well as after the second PCR; all agar-negative cell lines were also unambiguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has several outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.     

Simultaneous amplification of exons 18 to 21 of the EGFR gene using 5′ tailed primers and a two-stage protocol

Objective: Reduction of non-specific amplification and achievement of efficient amplification of multiple gene fragments under the same reaction condition is the basic goal of PCR diagnosis; however, this is often difficult. This study was conducted to establish a highly specific and effective amplification of the epidermal growth factor receptor (EGFR) gene's exons, 18–21, simultaneously. Methods: The 5′-tailed primers were synthesized by adding 10 to 20 bp of a non-specific sequence to the 5′-terminus of sequence-specific primers (tailless primers). The two-stage protocol consisted of 5–10 cycles of a conventional 3-step cycling, which was then followed by 30–35 cycles of two-step cycling. The exons 18–21 of EGFR gene were amplified in 28 non-small cell lung cancer (NSCLC) patients using an optimized PCR that combined 5′ tailed primers with a two-stage protocol. Results: The 5′ tailed primers exhibited a wider range of suitable annealing temperatures, similar range of primer concentration, similar sensitivity, specificity, and reproducibility, as well as a reduced, non-specific amplification compared with the corresponding tailless primers. The amplification of exons 18–21 of EGFR gene in NSCLC patients revealed that a combination of 5′ tailed primers with two-stage protocol (optimized PCR) had a similar PCR success rate (P = 0.873) but had significantly reduced non-specific amplification (P <0.001) compared to conventional PCR. Conclusion: 5′ tailed primers exhibited a wider range of suitable annealing temperatures and improved specificity compared with conventional PCR primers. An optimized PCR was established with 5′ tailed primers and a two-stage protocol to amplify exons 18–21 of the EGFR gene in NSCLC patients.     

Optimization of RAPD‐PCR conditions in cattle

Abstract

Random Amplified Polymorphic DNA polymerase chain reaction (RAPD‐PCR) is a fast and easy way of identifying DNA polymorphisms generated from several regions of the genome. This could expedite the process of identifying informative polymorphic markers that may be linked to important genes controlling economic traits. In cattle, failure to obtain consistent amplification patterns in RAPD‐PCR has been a cause for concern. This has been attributed to the fact that decamer primers that are used in RAPD‐PCR reactions are likely to amplify regions of DNA where the primer‐template base pairing has some degree of mismatch and that these mismatches fail to repeat from reaction to reaction. This paper describes the use of tricine buffer along with changes in reaction components and thermal cycling conditions that has yielded consistent and reproducible RAPD‐PCR amplifications using single primers and double primer combinations on bovine DNA.     

High GC Contents of Primer 5′-End Increases Reaction Efficiency in Polymerase Chain Reaction

Many studies have suggested that regulation of the polymerase chain reaction (PCR) is influenced by several factors. However, the understanding of reaction efficiency factors is not sufficient. Here we propose that high GC contents of primer 5′-end increases reaction efficiency in PCR. Using 71 primers (45 pairs), we analyzed factors that affect reaction efficiency, and statistically tested the correlation between the amplification signals and several factors. As a result, there were significant correlations between the amplification signals and the GC contents in the first 1～3 bps of primer 5′-end.     

四引物PCR扩增反应的单管SNP快速测定法

建立一种在单管中进行单核苷酸多型性 (SNP)快速测定的高效廉价方法 .以人ABCA1基因中的I82 3M为研究对象 ,设计 4种引物进行PCR扩增 ,其中两种引物用于扩增一段含有SNP位点的DNA片段 ,另两种引物为SNP位点特异性引物 ,4种引物在单管中同时进行PCR扩增反应 ,根据延伸产物的长度确定SNP的类型 .为提高SNP测定的特异性 ,在特异性引物的 3′端倒数第 3个碱基引入了一个人为错配碱基 ,使引物的错误延伸率显著降低 ,大大提高了SNP分析的准确性 .实验结果表明 ,所建立的方法简单 ,操作简便 ,可在单管中完成SNP的测定反应 .     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

基于荧光定量PCR扩增反应的SNP测定法

建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

Genome size and microsatellites: the effect of nuclear size on amplification potential.

Although the frequency of microsatellite DNA regions generally increases with increasing genome size, genome size has a negative effect on polymerase chain reaction (PCR) amplification. Thus, researchers developing sets of PCR primers, as is commonly done for microsatellite DNA regions, may encounter greater difficulty when working with species that have larger genomes. I investigated the effect of genome size on overall amplification success using data from nine different metazoan taxa. The proportion of primer sets that did not amplify PCR products was strongly and positively correlated with the haploid C value of the target species. Increasing genome size may affect amplification success negatively because of a decrease in target:nontarget DNA or by dilution of the available primer pool by nonspecific binding.     

从水稻Ac/Ds插入突变体扩增Ds侧翼序列的最适TAIL-PCR引物

温度非对称交互PCR(TAIL-PCR)技术已广泛应用于从多种生物体系克隆侧翼于已知序列的DNA片段的分子操作中,并极大地促进了反向遗传学研究。但是,可能由于不同物种间基因组大小和序列存在显差异,在采用该技术进行转座元件Ds水稻插入突变体鉴定过程中,常因TAIL-PCR反应的稳定性差而影响突变体筛选效率。有鉴于此,根据Ds核苷酸序列设计了分别对应或互补于Ds插入元件两端长度不同的12个特异引物组成32个组合,在大量预试验基础上与6个不同简并性(32～256)的随机简并引物分别组合进行TAIL-PCR反应,较系统地研究了引物特性对以水稻基因组DNA为模板的TAIL-PCR反应效率的影响。结果发现,第一反应采用长序列特异引物(36～40mer)可显提高扩增特异性,随机简并引物的简并度对反应的影响显。还选择出两个适于从水稻Ds插入突变体基因组高效扩增出Ds插入侧翼片段的最优特异引物组合和最适简并引物。应用本研究结果可显地提高TAIL-PCR技术筛选水稻插入突变体的效率。     

Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang.     

Phylogenetic Diversity of Natural Populations of Ammonia Oxidizers Investigated by Specific PCR Amplification

Abstract The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels. Received: 25 September 1995; Revised: 15 January 1996; Accepted: 20 February 1996     

盘羊肺炎支原体感染PCR 检测方法的建立与初步应用

羊支原体性肺炎,是由支原体引起的一类高度接触性传染病,又称羊传染性胸膜肺炎(Infections pleuropneumonia of sheep and goats),临床症状主要表现为高热、咳嗽、消瘦、     

简并引物在PCR扩增甘油—3—磷酸转酰酶基因中的应用

应用高简交引物,结合ＰＣＲ扩增技术从南瓜Ｃｕｃｕｒｂｉｔａ ｍｏｓｃｈａｔａ 总ＲＮＡ的逆转录产物中得到ＧＰＡＴ基因保守区。在ＰＣＲ反应中,简并引物之间的浓度配比是影响扩增效率的关键。用脱氧次黄苷替代简并引物中的高简交位中大幅度降低简并程度,显著提高扩增产物的特异性及扩增效率。     

Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5′ end of a gene, followed by denaturation and polyadenylation of its free 3′ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3′ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.