Successful Polymerase Chain Reaction Research Articles

Primer3—new capabilities and interfaces

Polymerase chain reaction (PCR) is a basic molecular biology technique with a multiplicity of uses, including deoxyribonucleic acid cloning and sequencing, functional analysis of genes, diagnosis of diseases, genotyping and discovery of genetic variants. Reliable primer design is crucial for successful PCR, and for over a decade, the open-source Primer3 software has been widely used for primer design, often in high-throughput genomics applications. It has also been incorporated into numerous publicly available software packages and web services. During this period, we have greatly expanded Primer3’s functionality. In this article, we describe Primer3’s current capabilities, emphasizing recent improvements. The most notable enhancements incorporate more accurate thermodynamic models in the primer design process, both to improve melting temperature prediction and to reduce the likelihood that primers will form hairpins or dimers. Additional enhancements include more precise control of primer placement—a change motivated partly by opportunities to use whole-genome sequences to improve primer specificity. We also added features to increase ease of use, including the ability to save and re-use parameter settings and the ability to require that individual primers not be used in more than one primer pair. We have made the core code more modular and provided cleaner programming interfaces to further ease integration with other software. These improvements position Primer3 for continued use with genome-scale data in the decade ahead.

DNA identification of formalin-fixed organs is affected by fixation time and type of fixatives: using the AmpFℓSTR®Identifiler®PCR Amplification Kit

Personal identification using DNA typing of formalin-fixed tissue is very important in the forensic sciences. However, few studies have been conducted to determine the detection limit of DNA typing of formalin fixation time in samples using the AmpFℓSTR(®) Identifiler(®) PCR Amplification Kit (Identifiler Kit). We collected samples from five cadavers submitted for forensic autopsies, and fixed them either in a 10% formalin solution, or in a 10% neutral-buffered formalin solution. The amount of template DNA for polymerase chain reaction (PCR) amplification and the detection limit of DNA typing for the Identifiler Kit were determined. When tissues were fixed in 10% formalin, 10 ng of DNA template was required for successful genotyping even after three-hour fixation and 100 ng was required after one-week fixation for PCR amplification. However, when tissues were fixed in 10% neutral-buffered formalin, the required amount of DNA template was 1 ng for a fixation time of three hours to three days and 125 ng for three months. Fixation time in neutral-buffered formalin was longer for successful PCR than that in formalin solution. Dropout was more common with increasing formalin fixation time. These results suggest that neutral-buffered formalin is preferred to formalin for fixation of tissues if they are to be subjected to DNA typing and that tissues fixed with neutral-buffered formalin can be used for DNA typing using the Identifiler Kit unless the fixation time exceeds one month.

Addressing challenges in non invasive capture-recapture based estimates of small populations: a pilot study on the Apennine brown bear

It is often difficult to determine optimal sampling design for non-invasive genetic sampling, especially when dealing with rare or elusive species depleted of genetic diversity. To address this problem, we ran a hair-snag pilot study on the remnant Apennine brown bear population. We used occupancy models to estimate the performance of an improved field protocol, a meta-analysis approach to indirectly model capture probability, and simulations to evaluate the effect of genotyping errors on the accuracy of capture-recapture population estimates. In spring 2007 we collected 70 bear hair samples in 15 5 × 5 km cells, using 5 10-day trapping sessions. Bear detectability was higher in 2007 than in a previous attempt on the same population in 2004, reflecting improved field protocols and sampling design. However, individual capture probability was 0.136 (95% CI = 0.120–0.152), still below the minimum requirements of capture-mark-recapture closed population models. We genotyped hair samples (n = 63) at 9 microsatellite loci, obtaining 94% Polymerase Chain Reaction success, and 13 bear genotypes. Estimated PIDsib was 0.00594, and per-genotype error rate was 0.13, corresponding to a 99% probability of correct individual identification. Simulation studies showed that the effect of non-corrected or filtered genetic errors on the accuracy of population estimates was negligible only when individual capture probability was >0.2. Our results underline how the interaction among field protocols, sampling strategies and genotyping errors may affect the accuracy of DNA-based estimates of small and genetically depleted populations, and warned us about the feasibility of a survey using only traditional hair-snag sampling. In this and similar cases, indications from pilot studies can provide cost-effective means to evaluate the efficiency of designed sampling and modelling procedures.

Multi-chamber PCR chip with simple liquid introduction utilizing the gas permeability of polydimethylsiloxane

On-chip polymerase chain reaction (PCR) is beginning to provide a viable alternative to conventional genetic profiling and amplification devices through minimal reagent use, short time, high detection resolution and potential high-throughput parallel testing of genetic materials. Despite the advantages, there are many challenges to overcome in accurate control and manipulation of fluid, circumventing bubble formation and inhibiting sample loss during PCR thermal cycling for successful PCR. In this research, gas permeability of polydimethylsiloxane (PDMS) was employed for liquid sample introduction into PDMS multi-chamber PCR chip, avoiding trapped bubbles in the reaction chambers. This method is simpler and more reliable compared to the other reported methods where integration of many complicated components, such as micropumps and micromixers on the chip for both sample loading and mixing are necessary. The sample evaporation and bubble formation on chip were controlled by using glycerol as a vapor pressure modifier. With this device, successful amplification of human β-Actin gene was demonstrated. This approach will be applicable in developing chip devices for multi-target sample amplification for diagnostic purposes.

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200bp.

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.

Purification of polymerase chain reaction (PCR)-amplifiable DNA from compost piles containing bovine mortalities

Livestock production systems utilize composting as a method of disposal of livestock mortalities, but there is limited information on the rate and extent of carcass decomposition. Detection of specific DNA fragments by PCR offers a method for investigating the degradation of carcasses and other biological materials during composting. However, the purity of extracted DNA is critical for successful PCR analysis. We applied a method to purify DNA from compost samples and have tested the method by analyzing bovine and plant DNA targets after 0, 4, and 12 month of composting. The concentration of organic matter from composted material posed a particular challenge in obtaining pure DNA for molecular analysis. Initially extracted DNA from composted piles at day 147 was discoloured, and PCR inhibitors prevented amplification of target plant or bovine gene fragments. Bovine serum albumin improved detection by PCR (25–50 μl final volume) through the removal of inhibitors, but only when concentrations of humic acids in extracted DNA were 1.0 ng μl −1 or less. Optimal purification of DNA from compost was achieved by chromatography using Sepharose 4B columns. The described DNA purification protocol enabled molecular monitoring of otherwise cryptic bovine and plant target genes throughout the composting process. The assay could likely be used to obtain PCR-amplifiable DNA that could be used for the detection of microbial pathogens in compost.

Study on the transmission of coconut Lethal Yellowing in Ghana.

Research on the Cape Saint Paul Wilt Disease (CSPWD) vector in Ghana began from 1990 (1990-1997; 2002-2004) and did not give convincing results. From July 2005, new test standards were applied: shading, daily collections and releasing of insects at the less hot hours and use of various sizes of cages and test plants. More than 70,000 Myndus. adiopodoumeensis were introduced in cage for 28 months (520 adults/seedling/month). Controls in polymerase chain reaction (PCR) on the five coconut of this Myndus cage and on 935 adults were always negative. The tests of transmission with M. adiopodoumeensis apparently not a vector of the disease were thus stopped. The phytoplasma of the CSPWD was identified by PCR in a coconut having received 4,380 Diostrombus (four species of Derbidae) 4 months after the beginning of the test. This coconut never presented symptom of the disease 28 months later and all the successive PCR were negative. Auchenorrhyncha collected by sweeping on the adventitious plants in and around the plot during the day were also tested without success. The hypothesis of a ground transmission was also taken into account because of the presence of scale insects and nematodes.

Study on the transmission of coconut Lethal Yellowing in Ghana

Research on the Cape Saint Paul Wilt Disease (CSPWD) vector in Ghana began from 1990 (1990-1997 ; 2002-2004) and did not give convincing results. From July 2005, new test standards were applied : shading, daily collections and releasing of insects at the less hot hours and use of various sizes of cages and test plants. More than 70,000 Myndus. adiopodoumeensis were introduced in cage for 28 months (520 adults/seedling/month). Controls in polymerase chain reaction (PCR) on the five coconut of this Myndus cage and on 935 adults were always negative. The tests of transmission with M. adiopodoumeensis apparently not a vector of the disease were thus stopped. The phytoplasma of the CSPWD was identified by PCR in a coconut having received 4,380 Diostrombus (four species of Derbidae) 4 months after the beginning of the test. This coconut never presented symptom of the disease 28 months later and all the successive PCR were negative. Auchenorrhyncha collected by sweeping on the adventitious plants in and around the plot during the day were also tested without success. The hypothesis of a ground transmission was also taken into account because of the presence of scale insects and nematodes.

PCR Primer Design

INTRODUCTIONThe objective of a polymerase chain reaction (PCR) is to amplify a specific DNA segment without any nonspecific by-products. In principle, each physical and chemical component of PCR can be modified to produce a potential increase in yield, specificity, or sensitivity. Yet the most critical parameter for successful PCR is optimal primer design. A poorly designed primer can result in little or no product, due to nonspecific amplification and/or primer-dimer formation leading to reaction failure, even when all the other parameters are properly optimized. This article provides general guidelines for PCR primer design, tips for development of primer pairs for more complex applications, and advice on the development of probes for real-time PCR.

The Effects of Tissue Fixation Alternatives on DNA Content

The aim of this study is to investigate the effects of different fixatives on DNA, and to evaluate the fixation options for molecular studies including polymerase chain reaction (PCR) and fluorescence in-situ hybridization (FISH). Three normal-looking colonic segments from surgical resections were used for tissue sampling. The full thickness of the colonic tissues (3 mm diameter) was sampled. Tissues were fixed in 70% ethanol, 10% neutral-buffered formalin, Hollande, B5, Bouin, and Zenker solutions for 1, 2, 5, 12, 24, and 48 hours, and processed and embedded in paraffin in a standard protocol. Quantitative measurements of the extracted DNA were carried out. DNA quality was tested by PCR for the human beta globin gene. Tissue sections were also tested for the availability of FISH, by using a Her-2/neu protocol. All fixation alternatives were found to be reasonable sources of DNA for molecular studies, and they enabled the successful PCR amplification of a housekeeping gene. DNA yields were predominantly over 1000 bp in 70% ethanol and 24-hour 10% neutral-buffered formalin fixations. As for B5 and Hollande, the DNA molecules obtained were almost all smaller than 100 bp. All tissues fixed in formalin, ethanol, and Hollande were found suitable for Her-2/neu visualization after standard FISH applications, but tissues fixed in Zenker, B5, and Bouin were not found suitable.

Development of a Polymerase Chain Reaction Test for Entamoeba invadens

Entamoeba invadens is a protozoal parasite of reptiles that causes colitis, abscesses of liver and other organs, and sometimes acute death. It is generally considered a commensal of chelonians but has also been implicated as a cause of colitis, diarrhea, and death in gopher (Gopherus polyphemus) and leopard (Geochelone pardalis) tortoises. Diagnosis of E. invadens is currently by detection of trophozoites and/or cysts upon direct fecal examination. However, definitive diagnosis of E. invadens has been difficult due to the very similar morphology of nonpathogenic Entamoeba spp., including E. ranarum, E. insolita, E. barreti, and E. terrapinae. Definitive speciation of Entamoeba spp. is important to avoid misdiagnosis or overtreatment for nonpathogenic protozoa. It is also important for consideration of mixed species reptile collections to avoid exposing snakes and lizards to E. invadens. In this study, we developed polymerase chain reaction (PCR) primers for E. invadens, E. ranarum, E. terrapinae, and E. insolita and conducted PCR amplification of purified DNA from cell cultures, as well as purified DNA from reptile stool samples with E. invadens trophozoites added. As a result of this study, a naturally occurring infection of E. invadens was confirmed in a giant South American river turtle (Podocnemis expansa). This study has developed successful PCR primers for four species of Entamoeba and demonstrates that PCR is a promising diagnostic tool for the definitive identification of E. invadens.

HotStart-IT ® : A Novel Hot Start PCR Method Based on Primer Sequestration

HotStart-IT ^® : A Novel Hot Start PCR Method Based on Primer Sequestration

Rapid and cost-effective detection of sequence-specific DNA by monitoring the electrochemical response of 2′-deoxyguanosine 5′-triphosphate in a PCR sample

This study describes a novel strategy for rapid and cost-effective detection of sequence-specific DNA based upon the essential utility of the polymerase chain reaction (PCR) and electrochemical technologies. A dramatic enhancement of the anodic peak current (i(pa)) and a visible decrease of overpotential towards free 2'-deoxyguanosine 5'-triphosphate (dGTP) could be realized on a glassy carbon electrode modified with short single-walled carbon nanotubes (S-SWNT/GCE). Thereby, the concentration of the free dGTP in the PCR sample mixture could be determined sensitively. The i(pa) of the free dGTP decreased remarkably after a successful PCR amplification owing to the participation of the free dGTP as one of the reactive substrates for the PCR products, namely dsDNA. Based upon this response change of the free dGTP before and after incorporation in PCR, a novel method aiming at detecting PCR results was established. One transgenic maize sample as a model was successfully detected by employing the specific sequences of 35S promoter from cauliflower mosaic virus (CaMV35S) gene and nopaline synthase (NOS) gene as markers. The result was in good accordance with that obtained with gel electrophoresis.

Micro–Rayleigh-Bénard convection polymerase chain reaction system

Polymerase chain reaction (PCR) is a molecular biological method for in vitro amplification of nucleic acids. Our objective was to design a micro-PCR system that included a Rayleigh-Bénard convection PCR chip, measurement circuits, and circuits to control the temperature. A Rayleigh-Bénard convection PCR chip was easily fabricated by using microelectromechanical system technology, and the sample solution could be put in to finish the completed PCR cycling within several minutes. The flow stream, velocity, and temperature profile in a micro-PCR system are important to achieve the successful PCR, but they are not easily observed with an experimental method. Thus, a CFDRC simulation of Rayleigh-Bénard convection PCR was undertaken to determine the above important parameters. The duration of one cycle, the extension time, and total duration of 25 cycles can be calculated to achieve the optimal design. Finally, using agarose-gel electrophoresis, we verified the practicability of this system. The comparison study of PCR experiments performed with a commercial PCR machine and our chips showed that our chips can greatly decrease the duration of the reaction. By comparing the simulation and PCR experiment results with varied designed sizes, a user can set the parameters and computational fluid dynamics results for optimal designs and decrease the total duration of future reactions.

Ageing and environmental factors affect PCR success in wolf (Canis lupus) excremental DNA samples

AbstractIndividual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf (Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples.

Biological and environmental degradation of gorilla hair and microsatellite amplification success

Naturally shed hairs are an important source of genetic material for both conservation and forensics but are notoriously poor sources of DNA. DNA degradation in hair roots is caused by apoptosis as part of the cycle of hair growth and by autolysis in decomposing animals. Shed hairs are additionally exposed to degenerative environmental processes. However, genetic studies rarely examine hair root morphologies or refer to root growth phases prior to analysis, and detailed knowledge of the rapidity of DNA degradation amongst shed hairs is lacking. We examined the effects of biological and environmental processes on western lowland gorilla (Gorilla gorilla gorilla Savage and Wyman) hair roots with respect to morphological characteristics and polymerase chain reaction (PCR) success at eight nuclear loci. Root type frequencies indicate that gorilla body hairs may exhibit a longer telogen phase than human head hairs. All plucked hair root types amplified more efficiently than shed hairs, and only 41% of shed hairs had root types considered suitable for genotyping. Telogen hairs from fresh nests were four-fold more useful for genotyping if the roots were associated with translucent epithelial tissue, and preselection of these root types doubled the overall data-yield to 58%. Nest age correlated with root morphology and PCR success, and PCR success was almost halved after 3 days of exposure. Finally, an association between postmortem interval, root morphology, and PCR success was observed that was consistent with postmortem changes reported in human head hairs.

A circular ferrofluid driven microchip for rapid polymerase chain reaction

In the past few years, much attention has been paid to the development of miniaturized polymerase chain reaction (PCR) devices. After a continuous flow (CF) PCR chip was introduced, several CFPCR systems employing various pumping mechanisms were reported. However, the use of pumps increases cost and imposes a high requirement on microchip bonding integrity due to the application of high pressure. Other significant limitations of CFPCR devices include the large footprint of the microchip and the fixed cycle number which is dictated by the channel layout. In this paper, we present a novel circular close-loop ferrofluid driven microchip for rapid PCR. A small ferrofluid plug, containing sub-domain magnetic particles in a liquid carrier, is driven by an external magnet along the circular microchannel, which in turn propels the PCR mixture through three temperature zones. Amplification of a 500 bp lambda DNA fragment has been demonstrated on the polymethyl methacrylate (PMMA) PCR microchip fabricated by CO(2) laser ablation and bonded by a low pressure, high temperature technique. Successful PCR was achieved in less than 4 min. Effects of cycle number and cycle time on PCR products were investigated. Using a magnet as the actuator eliminates the need for expensive pumps and provides advantages of low cost, small power consumption, low requirement on bonding strength and flexible number of PCR cycles. Furthermore, the microchip has a much simpler design and smaller footprint compared to the rectangular serpentine CFPCR devices. To demonstrate its application in forensics, a 16-loci short tandem repeat (STR) sample was successfully amplified using the PCR microchip.

La PCR dans la détection de l'ADN du virus de l'hépatite B : choix des amorces

Hepatitis B remains an important public health problem in the world; the agent causing this disease was characterized by its genetic variability. Hepatitis B surface antigen (HBs Ag) has been used for a long time for the indirect detection of the virus. Recently, the detection of the viral DNA by polymerase chain reaction (PCR) has been shown as a significant methodological progress for detecting the presence of viral nucleic acids even when these are found in small quantities in serum. This molecular diagnosis of the hepatitis B virus (HBV) infection based on PCR is confronted with problems of reproducibility and sensitivity and with difficulties of results interpretation. In this study, 13 primers specific and the primers consensus covering various regions of the hepatitis B virus genome were selected in order to perform an efficient and reproducible diagnostic test of HBV infection. The specificity and sensibility of PCR amplification were studied on sera of patients reached of chronic hepatitis B. Satisfactory results were obtained in the pre-S and X regions with respectively two successive PCR using the primers consensus or nested PCR using the couple Xa/Xb for the first amplification and the couple HBx1/HBx2 for the second. These results confirm that HBV DNA detection by PCR is a sensitive technique to detect the presence of HBV-DNA in the serum. It can be standardized to be apply in the currently diagnosis of HBV infection.

Ultrasonic Decalcification Offers New Perspectives for Rapid FISH, DNA, and RT-PCR Analysis in Bone Marrow Trephines

The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.