Controlled Microwave Heating Accelerates Rolling Circle Amplification

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.     

One-base excess adaptor ligation method for walking uncloned genomic DNA

This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5′-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5′-overhang base overlaps the corresponding 3′-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3′-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5′-terminal base was not consistent with the corresponding 3′-terminal base. Using this technique, the 3′- and 5′-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.     

利用微波-热效应进行限制性内切核酸酶反应的可行性验证

限制性内切酶催化的酶解反应是分子生物学实验中的常用技术.鉴于酶切反应时间长,有人提出采用微波释放的热效应催化限制性内切酶解反应,以期缩短酶解时间.但微波是否能代替传统酶切反应还需验证.本研究设计了一系列的酶切 试验,验证微波酶切是否有效.结果表明：短时间（2 min）微波炉高火处理未导致DNA降解及内切酶失活.对于质粒的单酶切和部分双酶切,短时间（2 min）微波炉处理可替代水浴加热完成酶解反应.对于部分双酶切体系,微波炉处理酶切不完全,不能替代温水浴.使用微波酶解需严格控制时间,切勿时间过长.     

RNA-templated DNA ligation for transcript analysis

Ligase-mediated gene detection has proven valuable for detection and precise distinction of DNA sequence variants. We have recently shown that T4 DNA ligase can also be used to distinguish single nucleotide variants of RNA sequences. Here we describe parameters that influence RNA-templated DNA ligation by T4 DNA ligase. The reaction proceeds much more slowly, requiring more enzyme, compared to ligation of the same oligonucleotides hybridized to the corresponding DNA sequence. The reaction is inhibited at high concentrations of ATP and NaCl and both magnesium and manganese ions can support the reaction. We define reaction conditions where 80% of RNA target molecules can template a diagnostic ligation reaction. Ligase-mediated RNA detection should provide a useful mechanism for sensitive and accurate detection and distinction of RNA sequence variants.     

Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases

A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Replication of bacteriophage T4 DNA in vitro. I. Basic properties of the system.

A new in vitro system for T4 DNA replication was developed by concentrating cell lysates on cellophane disks. The time course of [3H]dTTP incorporation into DNA by the system was separated into two phases: one was a very rapid incorporation which was terminated within 2 min (phase I reaction), and the other was a slow but continuous incorporation thereafter (phase II reaction). More than half of the phase I reaction product was Escherichia coli DNA, but the phase II reaction was mostly T4 DNA. Phase II reaction required four deoxyribonucleoside triphosphates, ATP, Mg2+, and KCl. 5-Hydroxymethyldeoxycytidine triphosphate was essential for the reaction and not substitutable by dCTP. The presence of KCN or NaN3 in the reaction mixture did not interfere with [3H]dTTP incorporation, but the addition of deoxyribonuclease completely degraded the system. Alkaline sucrose sedimentation analysis of phage II reaction product revealed that phase II reaction proceeded by the discontinuous mode of DNA replication as in vivo. After T4 infection, the activity for phase II reaction appeared in parallel with the activity of T4 phage DNA replication in vivo.     

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.     

Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

Microwave esterification of sunflower proteins in solvent-free conditions

The esterification of storage proteins from sunflower seeds renders these molecules more hydrophobic and increases internal plasticization, facilitating their use in the fabrication of materials by thermo-mechanical processes. The reaction was carried out in solvent-free conditions to simplify the process and to reduce costs. Optimization of esterification by the classical method, heating in a thermostat-regulated oil bath, resulted in 84% esterification for a reaction time of 4 h (amount of catalyst = 3.9 meq 5 N HCl/g protein, T = 90 degrees C). Microwave heating was then used to reduce the reaction time and the hydrolysis of protein chains. A similar level of esterification (89%) was obtained in 18 min (amount of catalyst = 3.4 meq 5 N HCl/g protein, microwave power (P) = 560 W). High yields were obtained with this method, demonstrating that this process limited hydrolysis.     

Telomere length measurement by a novel monochrome multiplex quantitative PCR method

The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R² = 0.844) than with our original singleplex method (R² = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R² = 0.91).     

Exonuclease Degradation of DNA Studied by Fluorescence Correlation Spectroscopy

Abstract

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and/or single-stranded DNA/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3′ to 5′ exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times. We give proof of (i) complete enzymatic degradation, (ii) retardation of complete enzymatic degradation by internally labelled Rhodamine-4-nucleotides and Cy5-nucleotides, respectively. Data evaluation by global analysis indicated first-order reaction kinetics with full-length DNA and free fluorescent nucleotides in the time window of measurements used.     

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography–tandem mass spectrometry coupled with microscale labeling

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH₄HCO₃ concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.     

Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase

Despite its unique ability to ligate single-stranded DNA molecules, T4 RNA ligase has so far seen little use in molecular biology due to long reaction times, modest yields, and apparent inability to promote ligation of long oligodeoxyribonucleotides. We describe here a set of reaction conditions which dramatically shorten the reaction time and give reproducible 40 to 60% ligation of DNA fragments of up to 40 bases in length. These improvements open promising new fields of application to T4 RNA ligase.     

Does DNA absorb microwave energy?

The microwave absorption of chicken erythrocytes and E. coli DNA aqueous solutions was studied in the 9–12-GHz frequency range by the method of variable thickness. At the same frequencies, fragments of sonicated erythrocyte DNA (average size about 500 base pairs) and of E. coli DNA treated with DNase (most of which were 800–2800 base pairs) were investigated. In neither case was any effect of enhanced microwave absorption by DNA observed. It was shown that an excess absorption of DNA solution falls within the 1% experimental error range, provided the conductivity contribution of 1% MgCl₂, required for DNase action, to the microwave absorption is taken into account.     

Novel enzymatic properties of DNA-Pt complexes

DNA-Pt complexes have shown novel enzymatic activity as a peroxidase similar to that of horseradish peroxidase in the colorimetric reaction with its substrate. The enzymatic activity of these complexes increased with increasing reaction time and pH in reaction solutions of DNA and K2[PtCl4]. This enhanced enzymatic activity was attributed to the increase in Pt conjugated to DNA in the complex. The enzymatic activity per unit mole of the DNA-Pt complex was significantly higher for complexes prepared with high molecular weight DNA because the enzymatic activity of the complex per repeat unit of DNA was almost constant for these complexes prepared under the same reaction conditions. All the DNA-Pt complexes in this study prepared with different DNA sequences (i.e., [A]20, [G]20, [C]20, [T]20, and [AG]10) exhibited peroxidase enzymatic activity. These complexes showed good thermal stability as compared to native horseradish peroxidase.     

一种快速高效获取丝状真菌PCR反应模板的方法

建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。     

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time. Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997     

Accelerated esterification of free fatty acid using pulsed microwaves

It was demonstrated that pulsed microwave irradiation is a more effective method to accelerate the esterification of free fatty acid with a heterogeneous catalyst than continuous microwave irradiation. A square-pulsed microwave with a 400 Hz repetition rate and a 10-20% duty cycle with the same energy as the continuous microwave were used in this study. The pulsed microwaves improved the esterification conversion from 39.9% to 66.1% after 15 min in comparison with the continuous microwave under the same reaction conditions. These results indicated that pulsed microwaves with repetitive strong power could enhance the efficiency of biodiesel production relative to the use of continuous microwave with mild power.