Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling

A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four different amplicons of 200, 300, 400, and 480 bp. However, the degree of amplification of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 degrees C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buffer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within +/-0.5 degrees C. The multiplex PCR approach will be further refined and tested for realization of an easily accessible alternative to a physical temperature measurement device in testing the performance of thermal cyclers for PCR.     

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.     

Thirty-cycle temperature optimization of a closed-cycle capillary PCR machine

The performance of a novel thermal cycler has been characterized in a 30-cycle PCR. The device consists of a microcapillary equipped with bidirectional pressure-driven flow and in situ optical position sensors. A 1-microL droplet of reaction mixture moves between three heat zones in a 1-mm i.d., oil-filled capillary using a multi-element scattered light detector and active feedback. The design permits time and number of cycles to be changed without hardware modification, unlike other flow-in-capillary PCR systems. Temperature optimization has been performed on the three PCR heat steps. The optimal denaturation temperature is 94 degrees C-96 degrees C, which is identical to commercial machines. The optimal extension temperature of 62 degrees C-66 degrees C is lower than reported for Taq DNA polymerase (70 degrees C-80 degrees C) because of the high enzyme concentration and/or the absence of detergent in the PCR mixture. The optimal annealing temperature seems to be the same as the optimal extension temperature. This is because extension occurs when the sample is inside of the annealing heat zone. Annealing takes place as the sample travels between heat zones. Device speed (23 minfor 30 cycles without time optimization) is competitive with other rapid PCR designs for efficiencies comparable to a commercial machine.     

In vitro amplification of DNA fragments greater than 10 kb.

The synthesis of a 10.9-kb DNA fragment from a bacteriophage lambda template was used in the search for conditions to extend the range for the polymerase chain reaction (PCR). Using the same primer sequences and conditions (denaturation at 94 degrees C, 1 min; annealing at 57 degrees C, 1 min; polymerization at 70 degrees C, 20 to 30 min) as published by W. Rychlik, W. J. Spencer, and R. E. Rhoads [(1990) Nucleic Acids Res. 18, 6409-6412], unsatisfactory results were obtained with AmpliTaq and native Taq polymerase (poor reproducibility, low product yield, nonspecific products), whereas Tub polymerase completely failed to amplify this fragment. Only after changes in the following parameters were reliable results obtained but only with Tub polymerase: A two-step PCR procedure with primer annealing and extension at 65 degrees C followed by DNA denaturation at 94 degrees C for 1.5 min was performed. The DNA fragment desired was specifically amplified when the enzyme concentration was reduced to 0.4 U/50 microliters and extension times as low as 4 to 12 min with an optimum at 8 min were used. A prolongation to 20 min or more resulted in an accumulation of unspecific products with a concomitant reduction in the yield of the fragment. Under the conditions described above it was also possible to amplify a DNA fragment even significantly longer (15.6 kb).     

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.     

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65 degrees C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65 degrees C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.     

Rapid detection of virulence <Emphasis Type="Italic">stx</Emphasis>2 gene of Enterohemorrhagic <Emphasis Type="Italic">Escherichia coli</Emphasis> using two-step ultra-rapid real-time PCR

A rapid detection method for Enterohemorrhagic Escherichia coli (EHEC), which has the virulent stx2 gene, was developed using a two-step, ultra-rapid real-time (URRT) PCR. URRT PCR was designed to detect the stx2 gene using a microchip-based, real-time PCR system, GenSpector TMC-1000, which only has a 6 μl total reaction volume with an extremely short denaturation step and combined annealing/extension step (1 and 3 s, respectively) for each cycle. Specific primers for the stx2 gene were designed to amplify a 100 bp region known for genetic stability among the various EHEC strains. Using the URRT PCR method, stx2 gene could be detected in 7 min 8 s including melting point (Tm) analysis. The detection limit for the stx2 gene for URRT-PCR was estimated to be 3 c.f.u./PCR with the amplification product having a consistent Tm of 85.2 ± 0.4°C. This method was tested for the various applications relevant to the different EHEC strains and was useful for the rapid detection of stx2-carrying EHEC strains.     

Amplification ratio control system for copy number variation genotyping

We describe a generic design for ratiometric analysis suitable for determination of copy number variation (CNV) class of a gene. Following two initial sequence-specific PCR priming cycles, both ends of both amplicons (one test and one reference) in a duplex reaction, are all primed by the same universal primer (UP). Following each amplification denaturation step, the UP target and its reverse complement (UP') in each strand form a hairpin. The bases immediately beyond the 3'-end of the UP and 5' of UP' are chosen such as not to base pair in the hairpin (otherwise priming is ablated). This hairpin creates a single constant environment for priming events and chaperones free 3'-ends of amplicon strands. The resultant 'amplification ratio control system' (ARCS) permits ratiometric representation of amplicons relative to the original template into PCR plateau phase. These advantages circumvent the need for real-time PCR for quantitation. Choice of different %(G+C) content for the target and reference amplicons allows liquid phase thermal melt discrimination and quantitation of amplicons. The design is generic, simple to set up and economical. Comparisons with real-time PCR and other techniques are made and CNV assays demonstrated for haptoglobin duplicon and 'chemokine (C-C motif) ligand 3-like 1' gene.     

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.     

A strategy to increase the specificity of Syber Green I qRT-PCR in hepatitis A detection

Various methods for the recovery and detection of HAV have been suggested, and molecular tests have recently provided an effective replacement for the traditional methods. Real-time RT-PCR technology offers many advantages over conventional RT-PCR in terms of rapidity and specificity. Most procedures are based on the TaqMan chemistry, but some researchers have used the SYBR Green I approach, which is less expensive and simpler to carry out. However the formation of primer-dimers needs to be distinguished from specific products through a melting curve analysis. This study focused on a strategy to increase the specificity of Syber Green I chemistry, thus nullifying the primer-dimers interference. To this end, forward and reverse primers were specially designed for hairpin loop formation, a strategy widely used to improve the specificity and the efficiency of PCR. Two different concentrations of primers were assayed (200 nM and 400 nM) in a one-step, real-time RT-PCR procedure, evaluating the specificity of the amplicons and the optimization of the real-time protocol. We demonstrated that this approach can increase the specificity of the Syber Green I qRT-PCR performance with a good reproducibility of the method. Because of the simplicity of the assay and the lower costs involved, this procedure could be a valid alternative to HAV monitoring from environmental matrices.     

Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.     

Fast DNA isolation and PCR protocols for detection of methicillin-resistant staphylococci

A rapid method that included simple boiling DNA extraction followed by a fast polymerase chain reaction (PCR) cycling protocol designed to detect mecA, which characterizes methicillin-resistant Staphylococcus aureus (MRSA), was performed. Briefly, the PCR cycling protocol consisted of pre-denaturation at 95 °C for 30 s, 30 cycles of denaturation at 94 °C for 2 s, annealing at 52 °C for 5 s, extension for 10 s, and final extension at 72 °C for 1 min. A good level of reliability of the method was verified. The study has shown that the method described here represents a rapid and accurate DNA extraction and PCR-based identification system of MRSA, thus allowing clinicians to make early identification and early implementation of control measures.     

Adaptor long-range PCR procedure for clone-specific characterization and chromosomal localization

An efficient adaptor long-range PCR (ALR-PCR) procedure was developed to detect genomic rearrangements in high-plasticity genomic regions between closely related strains of bacteria. The method was precisely optimized using a combination of high-speed experimental steps for the chromosomal localization and elucidation of deletions, inversions, duplications, or inserted sequences within a clone-specific flanking region. The advantages of this strategy are: (i) ready-to-use polymerase mixtures and Master mix (ready-to-use reaction mixtures with polymerase MasterAmp and buffer 2x Premix 4); (ii) a 5-min ligation procedure; (iii) rapid purification of DNA digests; (iv) optimized DNA template concentration protocol to avoid nonspecific amplification and high backgrounds; (v) long-range PCR protocol to obtain at least 9.6 kb single PCR products; (vi) two-step PCR cycling with the same annealing and extension temperature at 68 degrees C; (vii) simple design of the adaptors according to the preferred restriction endonuclease enzyme; and (viii) simple technology and equipment required. The application of this method for a tester-specific suppressive subtractive hybridization (SSH) clone of Brucella melitensis 16M revealed an 837-bp deletion and a 7255-bp DNA transfer from one chromosomal location to another for Brucella abortus 2308 used as a driver.     

Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion

Background

Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens.

Methods

Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature (T_c) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion.

Results

Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined.

Conclusions

TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.     

聚合酶链式反应热流变化的DSC实验研究

在PCR每个循环中,目的基因在DNA聚合酶的催化作用下实现快速扩增,同时伴随着化学键的断裂和生成,而不同循环数的扩增效率不同,引起的热现象也不同。实验通过差示扫描量技术,以HBV为PCR扩增体系,分别研究了变性、退火和延伸阶段的热焓及其随循环数的变化,通过分析得出：变性阶段是放热过程,第17个循环放热量达到最大,退火和延伸阶段是吸热过程;3个阶段的热焓随循环数增加都发生明显的变化,其中变性阶段的热流变化最关键。     

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of F?rster resonance energy transfer probes in 5′-nuclease assays

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5′-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem–loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease activity of Taq. The stem–loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. It was found that at a certain length of these 5′-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology’s real-time performance was compared to a number of conventional assays including Taqman, 3′-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay’s cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

Rapid differentiation of Staphylococcus aureus from staphylococcal species by arbitrarily primed-polymerase chain reaction

An arbitrarily primed-polymerase chain reaction (AP-PCR) method was optimized to differentiate Staphylococcus aureus from other staphylococcal species, using DNA from crude cell extract. From the different assays carried out, the best resolution of the band patterns was obtained when the reaction mixture contained 200 micromol l(-1) dNTPs, 200 ng primer, 1 U Taq DNA polymerase and 3 mmol l(-1) MgCl2 and the amplification conditions were: initial denaturation of 94 degrees C for 1 min, primer annealing of 30 degrees C for 1.5 min, DNA extension at 55 degrees C for 5 min and final extension at 55 degrees C for 5 min. The results of the characterization of the staphylococcal isolates by AP-PCR are in accordance with those of the biochemical identification by the API Staph System, time of analysis of the AP-PCR being only 6-7 h. Thus, this technique could be a useful method for microbial quality assurance.