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DNA-based procedures are becoming increasingly common within the analytical laboratory where the polymerase chain reaction (PCR) has become an indispensable technique. Developed in 1985 by Kary B. Mullis, PCR revolutionized the way that deoxyribonucleic acid (DNA) could be copied. Mullis's invention allowed researchers to make millions of copies of a selected DNA region within hours. Today, PCR can take minutes. PCR has widespread analytical applications in the food, environmental, medical and forensic fields. This Technical Brief covers the basics of PCR and some of its variations.

Motivation Polymerase chain reaction (PCR) has been a revolutionary biomedical advancement. However, for PCR to be appropriately used, one must spend a significant amount of effort on PCR primer design. Carefully designed PCR primers not only increase sensitivity and specificity, but also decrease effort spent on experimental optimization. Computer software removes the human element by performing and automating the complex and rigorous calculations required in PCR primer design. Classification and review of the available software options and their capabilities should be a valuable resource for any PCR application. Results This article focuses on currently available free PCR primer design software and their major functions (https://pcrprimerdesign.github.io/). The software are classified according to their PCR applications, such as Sanger sequencing, reverse transcription quantitative PCR, single nucleotide polymorphism detection, splicing variant detection, methylation detection, microsatellite detection, multiplex PCR and targeted next generation sequencing, and conserved/degenerate primers to clone orthologous genes from related species, new gene family members in the same species, or to detect a group of related pathogens. Each software is summarized to provide a technical review of their capabilities and utilities.

Premise Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time-consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high-quality DNA suitable for downstream PCR applications. Methods and results We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven-dried, acetone-fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone-fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real-time PCR assays. Conclusions The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real-time assays. This method is simple, fast, and cost-effective, and is a reliable tool for extracting high-quality DNA from plant material containing PCR inhibitors.

Molecular technologies based on PCR have been widely used in many biological analysis fields, especially for genetic analysis and DNA barcoding. In this study, a rapid DNA lysis liquid was formulated without any purification step from fresh and processed meat, suitable for conventional PCR amplification. Three different lysis liquid formulas were designed for selection and further optimization for direct PCR and absorbance spectra; DNA concentration and performance in PCR were used to assess the effect of each formula. The results indicated that the formula containing NaOH, EDTA, SDS, Tween 20, and Tris-HCl achieved the best results, and the optimized formula met the need of practical PCR applications. The protocol provided a rapid lysis buffer for DNA replication from any meat samples. The performance of the final formula resulted in high DNA lysis efficiencies for all the tested meat samples and the PCR amplification efficiencies were similar to isolated DNA template using a commercial kit. The whole process can be completed in 30 min. Therefore, this study provides a simple, alternative, cost-effective fast solution for meat molecular analysis based on DNA.

Point mutations in DNA are useful biomarkers that can provide critical classification of disease for accurate diagnosis and to inform clinical decisions. Conventional approaches to detect point mutations are usually based on technologies such as real-time polymerase chain reaction (PCR) or DNA sequencing, which are typically slow and require expensive lab-based equipment. While rapid isothermal strategies such as recombinase polymerase amplification (RPA) have been proposed, they tend to suffer from poor specificity in discriminating point mutations. Herein, we describe a novel strategy that enabled exquisite point mutation discrimination with isothermal DNA amplification, using mismatched primers in conjunction with a two-round enrichment process. As a proof of concept, the method was applied to the rapid and specific identification of drug-resistant Mycobacterium tuberculosis using RPA under specific conditions. The assay requires just picogram levels of genomic DNA input, is sensitive and specific enough to detect 10% point mutation loading, and can discriminate between closely related mutant variants within 30 min. The assay was subsequently adapted onto a low-cost 3D-printed isothermal device with real-time analysis capabilities to demonstrate a potential point-of-care application. Finally, the generic applicability of the strategy was shown by detecting three other clinically important cancer-associated point mutations. We believe that our assay shows potential in a broad range of healthcare screening processes for detecting and categorizing disease phenotypes at the point of care, thus reducing unnecessary therapy and cost in these contexts.

Classical analytical biotechnology typically requires hundreds of thousands of molecules as substrates for analysis. In the case of the deoxyribonucleic acid (DNA), the in vivo molecular cloning has been used to generate virtually unlimited number of molecules for downstream processing. Yet, that is usually a labor-intensive task. These limitations can be overcome with in vitro amplification strategies, which are in fact cloning approaches from the functional (amplification) point of view. The keystone of these methodologies is the exponential amplification of DNA, allowing to generate millions of molecules from a single molecule, in just a few minutes. The polymerase chain reaction (PCR) is the most popular in vitro nucleic-acid amplification methodology. In fact, it can be said that the PCR has represented an inflexion point in science and technology, changing the way biotechnology is done by anyone working with organic entities (viroids, virusoids, prokaryotes and eukaryotes), parts of them or derived products, both current and ancient ones. To carry out optimized PCR amplifications, some mathematics, thermodynamics and bioinformatics should be taken into account. The human factor is also needed most of the times if optimal PCR performance is required. That is why the PCR design process is indeed an art, where the manual optimization of the researcher may make the difference and determine the success of a PCR amplification experiment. This chapter reviews the relevance of PCR in biotechnology from a methodological and applied point of view, highlighting the optimization process, as well as the troubleshooting of the technology, which has a significant impact in the biomedical sciences.

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

The polymerase chain reaction (PCR) is a sought-after nucleic acid amplification technique used in the detection of several diseases. However, one of the main limitations of this and other nucleic acid amplification assays is the complexity, size, maintenance, and cost of their operational instrumentation. This limits the use of PCR applications in settings that cannot afford the instruments but that may have access to basic electrical, electronic, and optical components and the expertise to build them. To provide a more accessible platform, we developed a low-cost, palm-size, and portable instrument to perform real-time PCR (qPCR). The thermocycler leverages a copper-sheathed power resistor and a computer fan, in tandem with basic electronic components controlled from a single-board computer. The instrument incorporates a 3D-printed chassis and a custom-made fluorescence optical setup based on a CMOS camera and a blue LED. Results are displayed in real-time on a tablet. We also fabricated simple acrylic microdevices consisting of four wells (2 μL in volume each) where PCR reactions take place. To test our instrument, we performed qPCR on a series of cDNA dilutions spanning 4 orders of magnitude, achieving similar limits of detection as those achieved by a benchtop thermocycler. We envision our instrument being utilized to enable routine monitoring and diagnosis of certain diseases in low-resource areas.

DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

Success in molecular biology techniques involving hybridization such as PCR and non-PCR-based techniques depends on annealing temperature prediction which derived itself from the melting temperature (T m ) of primers with a minimal error probability. Such calculation should be derived from the comparison of existing methods in a large benchmark set of sequences, as it was the case in this study. In the present study 22 primer design tools were compared based on the deviation of the predicted T m values of 158 primers using software versus the experimentally determined T m values. A paired t-test (False discovery rate (FDR) corrected P-values) and mean square deviation (MSD) were applied to assess all software packages. A significant variation was observed for the T m values of primers calculated by different tools in comparison with optimal experimental condition, which could end up causing wide error in amplification reactions. Based on FDR and MSD criteria, Primer3 Plus and Primer-BLAST performed the best prediction of T m . We practically aimed to provide an easy, safe and robust application of the current available T m predictor tools for all researchers especially for premiers to help them increase their success in different PCR and real-time PCR applications.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

Taking into account the limits of current genotyping methodologies, we have established a versatile direct PCR method on intact microtissue samples without prior DNA isolation. A simple and standard protocol was developed and validated on a wide range of living organisms including bacterial and fungal strains, plant species and human samples. This allows reliable amplification of target genomic DNA fragment directly from source material using minimal amount of tissue which makes DNA purification irrelevant for a number of biological applications. The direct PCR technique established here represents an excellent alternative to traditional amplification methods used for real-time detection. Since this approach was efficiently and universally applied for high-throughput molecular screening, its implementation will offer new insights for several investigations in human health, biomedical diagnosis, plant biotechnology, as well as in applied environmental and food microbiology.

Researchers have gained a deeper understanding of DNA-based encryption and its effectiveness in enhancing information security in recent years. However, there are many theoretical and technical issues about DNA-based encryption that need to be addressed before it can be effectively used in the field of security. Currently, the most popular DNA-based encryption schemes are based on traditional cryptography and the integration of existing DNA technology. These schemes are not completely based on DNA computing and biotechnology. Herein, as inspired by nature, encryption based on DNA has been developed, which is, in turn, based on two fundamental biological axioms about DNA sequencing: 1) DNA sequencing is difficult under the conditions of not knowing the correct sequencing primers and probes, and 2) without knowing the correct probe, it is difficult to decipher precisely and sequence the information of unknown and mixed DNA/peptide nucleic acid (PNA) probes, which only differ in nucleotide sequence, arranged on DNA chips (microarrays). In essence, when creating DNA-based encryption by means of biological technologies, such as DNA chips and polymerase chain reaction (PCR) amplification, the encryption method discussed herein cannot be decrypted, unless the DNA/PNA probe or PCR amplification is known. The biological analysis, mathematical analysis, and simulation results demonstrate the feasibility of the method, which provides much stronger security and reliability than that of traditional encryption methods.

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

The polymerase chain reaction (PCR) is a technology used to exponentially amplify a short, specific DNA region of interest, generating thousands to millions of copies of this specific region, all with a similar sequence and length. It is one of the few techniques that has undoubtedly revolutionized the field of molecular biology and diagnostics. Since the invention of the classical end-point PCR in 1986, the technique has gone through a revolutionary period, with a boost in 1992 due to the invention of real-time or quantitative PCR (qPCR), which allowed for monitoring the PCR reaction in real-time, thereby making the quantification of nucleic acids much more simple, accurate, and sensitive. It is a key technology that is present on almost every bench in a research or diagnostic laboratory. In this chapter we will give an overview of the qPCR technique, starting from its invention, describing the different methodologies and important factors to be taken into account when optimizing and using this widespread nucleic acid-based technique.

a b s t r a c t PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

Accurate identification of fungal pathogens using a sequence-based approach requires an extraction method that yields template DNA pure enough for polymerase chain reaction (PCR) or other types of amplification. Therefore, the objective of this study was to develop and standardise a rapid, inexpensive DNA extraction protocol applicable to the major fungal phyla, which would yield sufficient template DNA pure enough for PCR and sequencing. A total of 519 clinical and culture collection strains, comprised of both yeast and filamentous fungi, were prepared using our extraction method to determine its applicability for PCR, which targeted the ITS and D1/D2 regions in a single PCR amplicon. All templates were successfully amplified and found to yield the correct strain identification when sequenced. This protocol could be completed in approximately 30 min and utilised a combination of physical and chemical extraction methods but did not require organic solvents nor ethanol precipitation. The method reduces the number of tube manipulations and yielded suitable template DNA for PCR amplification from all phyla that were tested.

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280 nm and 260/230 nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows.

Genetic material from large patient cohorts is increasingly central to translational genetic research. However, patient blood samples are a finite resource and their supply and storage are often dictated by clinical and not research protocols. Our experience supports difficulty in amplifying DNA from blood stored in herparin; a scenario that other researchers may have or will encounter. This technical note describes a number of simple steps that enable successful PCR amplification. DNA was extracted using the Illustra Nucleon Genomic DNA Extraction Kit. PCR amplification was attempted using a number of commercially available PCR mastermixes. PCR DNA amplification failed using ReddyMix™ PCR Master Mix, Thermo-Start® (Thermo Scientific Inc. US) and ZymoTaq™ (Zymo research, US) PCR mastermixes, as demonstrated absence of products on gel electrophoresis. However, using the Invitrogen™ (Thermo Scientific Inc., US) Platinum® Taq DNA Polymerase, PCR products were identified on a 1% agarose gel for all samples. PCR products were cleaned with ExoSAP-IT® (Affymetrix Inc., US) and a sequencing reaction undertaken using a standard Big Dye protocol. Subsequent genotyping was successful for all samples for alleles at the CDH1 locus. From our experience a standard phenol/chloroform purification and using the Invitrogen™ Platinum® Taq has enabled the amplification of whole blood samples taken into lithium heparin and stored frozen for up to a month. This simple method may enable investigators to utilise blood taken in lithium heparin for DNA extraction and amplification.

In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products.

Oral streptococci represent about 20% of the total oral bacteria, so if it is possible to detect the presence of oral specific bacteria from a forensic specimen by Polymerase chain reaction, this could be used to verify the presence of saliva. Aim of this study is detection of Streptococcus salivarius which is one of the most common streptococci in oral bacteria and Streptococcus mutans which is common in cases of dental caries in various body fluids and skin swabs and assessment of which one of both organisms is more reliable in saliva identification, cross sectional study on Egypt population. Negative control samples (15 samples) were taken from various body fluids (urine, semen) and skin swabs. Mock forensic samples (85 samples) included fresh saliva, saliva, cotton fabrics contaminated with saliva, cigarette butts, bitten apple and semen mixed with saliva samples). DNA extraction was done using DNeasy blood and tissue kit (Qiagen, Tokyo, Japan). Polymerase chain reaction was done for DNA amplification using Polymerase chain reaction master mix then gel electrophoresis was done for samples qualification. Control bacteria were S. salivarius and Streptococcus mutans. Streptococcus salivarius was detected in 83.5% of all saliva contained samples and S. mutans was detected in 67% of saliva contained samples. Both bacteria were not detected in other body fluids and skin swabs, so S. salivarius is more reliable in saliva identification as well as differentiating it from other body fluids. Polymerase chain reaction is valuable in detection of saliva by detecting S. salivarius.

DNA is a typical organic compound with marked differences from other chemicals and biopolymers because DNA can be amplified by the enzyme polymerase. DNA can be, in principle, amplified from a single copy by the polymerase chain reaction (PCR). In this review, we focus our attention on the chemistry of PCR primers. Because PCR is basic technology in biology research fields, we sometimes use chemically labeled primers without any awareness of the chemistry they leave behind. We would like to emphasize that chemically labeled primers contain a lot of potential for different chemistry ideas and much study is still necessary to advance PCR for single-nucleotide polymorphism (SNP) typing, genetic diagnosis, and other fields. Two categories of primers, affinity-capture primers and signaling primers, are discussed from the viewpoints of their chemical concepts and applications. Affinity-capture primers are used for purification, isolation, and manipulation of PCR products by high specificity and affinity to the cognate molecules by moleculemolecule interactions, whereas signaling primers report the hybridization and/or progress of PCR amplification by a signal change, in most cases by a fluorescence change. The content of this review may be useful for a better understanding of the chemistry of PCR primers and, more importantly, for the invention of novel PCR chemistry.

Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

Nucleic acid detection methods have been rapidly evolving and play an important role in viral detection and quantification. One technology which emerged in the 1990s, polymerase chain reaction, better known as PCR, has established itself as a primary tool for molecular biology. In fact, this technology has been so widely adopted in the clinical virology laboratory it has, in many cases, completely replaced culture. At its core, PCR is a straightforward chemical reaction whereby one strand of template DNA is exponentially amplified. This chapter will give an overview of different PCR technologies available, as well as the strength and weaknesses of each. For a more in-depth review of their applications in the clinical laboratory, the reader is directed to the appropriate chapter(s) elsewhere in this text.

The polymerase chain reaction (PCR) is a very useful method for increasing the amount of DNA present in a sample to a larger amount accessible for analysis. PCR is modified in a wide range of methods for a large number of biomedical and forensic uses. The general method is described and its application to forensic biological science as well commonly encountered challenges with the PCR assay. Keywords: DNA; amplification; profiling; analysis; SGMplus; identifiler; PowerPlex

With the advent of molecular-based methods to detect the presence of microbial pathogens, technologies such as the polymerase chain reaction (PCR) and whole genome sequencing have revolutionized the field of food microbiology in ensuring that food products are pathogen free. In addition, these tests can also be applied to strain-specific identification, thus serving as an important tool for differentiating and identifying strains used in food production and as probiotics. The basis of PCR methods is the amplification of select sites in either DNA or RNA molecules such that the level of sensitivity is as low as one microbial cell or virus particle. Since the first days that PCR was rolled out and its potential as a powerful tool for microbial diagnostics was recognized, improvements in its chemistry and the machines that perform these reactions have been impressive. Real-time PCR, which enables concurrent amplification of nucleic acid target sites and the data readout indicating the status of the reaction, has reduced the time of analysis from 2 to 4 h to less than 1 h. Whole genome sequencing can be applied to isolated colonies as well as a nonconventional means as a culture-free detection and identification method. Improvements in these technologies have such potential that the detection and identification of all known microbial pathogens can be accomplished in one reaction. Many different types of molecular-based assays are described in this article.

The requirement to monitor the presence of genetically modified organisms (GMOs) has generated an increasing demand for reliable, rapid, and time and cost-effective detection methods. Nowadays, the most reliable detection mean is the DNA-based polymerase chain reaction (PCR). According to the different levels of detection requirements, there are usually four strategies: screening PCR, gene-specific PCR, construct-specific PCR, and event-specific PCR. Event-specific PCR is widely used in GMO detection due to its excellent specificity and accuracy, and most latter methods are developed based on it. As the demand increases, the detection methods undergo continuous improvement and innovation, such as: real-time quantitative PCR, multiplex PCR, universal primer multiplex PCR, multiplex ligation dependent probe amplification, isothermal PCR, and so on. The technology and instruments update and improve constantly, and there are novel methods undergoing research; these detection techniques would not only fulfil the demands of the governmental supervision but would also guarantee a safe food consumption environment for the consumer.

The polymerase chain reaction (PCR) is a technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing and extension. PCR is useful in the investigation and diagnosis of a growing number of diseases. PCR is also used in forensics laboratories. PCR can identify genes that have been implicated in the development of cancer. The present paper is an attempt to review basics of PCR in relation to its methods, application and use.

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Researchers face a significant problem in PCR amplification of DNA fragments with high GC contents. Analysis of these regions is of importance since many regulatory regions of different genes and their first exons are GC-rich. There are a large number of protocols for amplification of GC-rich DNA, some of which perform well but are costly. Most of the economical protocols fail to perform consistently, especially on products with >80% GC contents and a size of >300 base pairs. One of these protocols requires multiple additions of DNA polymerase during thermal cycling which therefore rules out its utility if a large number of samples have to be amplified. We have established a method for simultaneous amplification of specific PCR products from a large number of human DNA samples using general laboratory reagents. These amplicons have GC contents ranging from 65–85% and sizes up to 870 base pairs. The protocol uses a PCR buffer containing co-solvents including 2-mercaptoethanol and bovine serum albumin for amplification of DNA. A specific thermal cycling profile is also used which incorporates a high annealing temperature in the first 7 cycles of the reactions. The PCR products are suitable for different molecular biology applications including sequencing.

Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950-1050 µg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in1 h. Pyrosequencing results show the assay to be extremely (99%) sensitive, and spike-back experiments demonstrated a detection threshold of10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

In this paper, we report the construction of a polymerase chain reaction (PCR) device for fast amplification and detection of DNA. This device consists of an interchangeable PCR chamber, a temperature control component as well as an optical detection system. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 μl, while the heating and cooling rate was as fast as 12.7°C/second ensuring that the total time needed of only 25 min to complete the 35 cycle PCR amplification. An optimized PCR with two-temperature approach for denaturing and annealing (Td and Ta) of DNA was also formulated with the PCR chip, with which the amplification of male-specific sex determining region Y (SRY) gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system.

The two main molecules that are suitable for bacterial broad-range PCR are the 16S rRNA gene, consisting of approximately 1,540 bp (in Escherichia coli, 1,542 bp), and the 23S rRNA gene, consisting of approximately 2,900 bp (in E. coli, 2,904 bp). While the impact of this appears to be somewhat smaller for diagnostic broad-range PCR in cases of suspected monomicrobial infections, it can be dramatic for polymicrobial infections, microbial flora studies, or environmental studies, where certain bacterial taxa may become significantly over- or under-represented after PCR amplification. In addition, the inherent nonselective nature of broad-range PCR makes it susceptible to minute amounts of any bacterial DNA that might be encountered along the various steps of testing. The classical way to detect and identify bacteria by broad-range PCR is via visualization of PCR products by standard gel electrophoresis, followed by sequencing, preferably for both DNA strands of the products. Broad-range PCR offers two potential benefits: it lacks selectivity for particular groups of bacteria, and it can detect as well as identify culture-resistant, fastidious, damaged, and slow-growing microorganisms. Bacterial broad-range PCR has also attracted interest in transfusion medicine, in order to assess blood products for bacterial contamination. Conducting broad-range PCR analysis at a level of high analytical and clinical sensitivity is a complex task and remains one of the most difficult and challenging PCR applications. Sequence-based identification of positive diagnostic broad-range PCR products is generally advisable, even when real-time technology is used.

Polymerase chain reaction (PCR) provides a foundation for simple sequence repeat molecular marker-assisted selection (SSR MAS) in soybean. This PCR system and its various conditions have been optimized by many researchers. However, current research on the optimization of the PCR system focuses on double-primer PCR products. We compared single- and double-SSR primer PCR products from 50 soybean samples and found that the use of single-PCR primers in the reaction system can lead to amplified fragments of portions of the SSR primers in the PCR process, resulting in both false-positives and fragment impurity of double-primer PCR amplification, inconvenient for subsequent analysis. We used "single-primer PCR correction" to eliminate interference caused by single-primer nonspecific PCR amplification and improve PCR quality. Using this method, the precision and success rates of SSR MAS in soybean can be increased.

Microbes are abundant in marine and terrestrial ecosystems. However, the available evidence indicates that most microbial species in the environment cannot be cultivated. Accordingly, methodologies for characterizing genomes in environmental samples directly without cultivation have become foremost in importance. In this study, experimental conditions using PCR amplification to characterize such genomes were investigated with respect to the detection of target sequences present at low frequencies. Amplification of the isoleucyl-tRNA synthetase (IleRS) gene in Methanopyrus isolates was optimized regarding the choice of DNA polymerase, target sequence size, PCR primer size and the use of degenerate primers. Detection of IleRS was most sensitive when the target sequence size was about 1 kb. Among KOD, Taq and Pfu DNA polymerases, Pfu displayed the lowest efficiency, while KOD and Taq showed similar efficiency. Primers no shorter than 18mers gave satisfactory results. In addition, PCR bias caused by primer degeneracy could be alleviated by varying the balance between different added primers. Employing a combination of Taq DNA polymerase, a target DNA sequence approximately 1 kb in size, and primers of 20mer in length, PCR amplification of the IleRS gene could be achieved with as little as 2 pg pure isolated DNA template.

For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation.

Heteroduplex formation is known to occur during mixed-template polymerase chain reaction (PCR) using universal primers, and may represent a serious problem in several PCR-based analyses. A common way to eliminate heteroduplex formation is to use reconditioning PCR. Because we detected that reconditioning PCR was not always sufficient to prevent heteroduplex formation, we focused on developing methods for the elimination of heteroduplexes during PCR. We detected that the heteroduplex to homoduplex ratio can be decreased by the addition of Taq polymerase and by a decrease in the number of PCR cycles. An appropriate combination of both of these approaches can be a method generally applicable to decrease the formation of heteroduplexes.

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. our results show that single mismatches instigate a broad variety of effects, ranging from minor (7.0 cycle threshold, eg, A-A, C-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches. (J Mol Diagn 2010,12:109-117; DOI. 10.2353/jmoldx.2010.090035)

The purpose of this study has been to demonstrate the possibilities of uniform heating of a cavity, with great accuracy, by means of an integrated resistor built with the same dimensions as the cavity, i.e., with a high level of integration. For application purposes, a compact resistor allows increasing the number of cavities in which temperature can be independently controlled on the same substrate, which can prove critical for high-throughput screening applications. Potential applications lie in the field of biology or chemistry. In order to achieve the desired result, an optimization procedure was performed on the shape of the resistor. The heater size reduction enables a high level of integration with a reduced heating source surface area. Resistor shape has been optimized to reduce the influence of boundary effects, using improvements introduced in genetic algorithms. An experimental validation of the temperature profile inside the cavity has been carried out using a dye whose fluorescence depends on temperature, i.e., Rhodamine B, it will be shown herein that the optimized resistor allows for temperature cycling, e.g., for PCR applications.

The amplification of small amounts of nucleic acids via PCR (Mullis and Faloona, 1985) has undergone a tremendous development in biology, biochemistry, clinical diagnosis, and related fields. The typical three step reaction consisting of heat denaturation, primer annealing, and primer elongation together with the advanced technology in the instrumentation of thermal cyclers has made the reaction simple and fast. The idea to use a heat stable DNA polymerase, primers, and dNTPs in order to amplify double stranded DNA molecules in an exponential manner is not the only way to produce large amounts of nucleic acids starting from a few molecules only. This paper will give an overview of some alternatives and their applications.

This chapter describes the quantitative molecular techniques that serve as the basis of viral load assays, and discusses key issues and important variables that affect assay performance. Real-time methods offer the advantages of increased dynamic range, simplicity, reduced analysis time, and diminished risk of contamination. Although polymerase chain reaction (PCR) is the best developed and most widely used nucleic acid amplification strategy, other strategies have been developed and several serve as the basis of quantitative assays for viral nucleic acids. Quantitative assays based on nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA), and hybrid capture are commercially available. The specificity of real-time PCR can also be increased by including hybridization probes in the reaction mixture. NASBA is one of several transcription-based amplification methods that amplify RNA targets. Hybrid capture assays (HCAs) employ a signal amplification technology that can be applied to the detection and quantitation of DNA or RNA target molecules without amplification. Prior to the development of quantitative molecular techniques, laboratories were limited to cumbersome culture methods or insensitive antigen detection methods to determine viral load. Each of the quantitative molecular techniques has particular strengths and limitations that are inherent in the underlying nucleic acid amplification strategies. A thorough understanding of the key performance issues and features of the available quantitative molecular techniques is essential to the practice of modern clinical virology.

Mitochondrial dysfunction has reported in several diseases including diabetes, cancer, skeletal muscle disorders and neurodegenerative diseases such as Wolfram syndrome. Several different methods have evolved to study mtDNA damage including Southern blotting, 8-oxoG damage, or a comprehensive scanning of the mitochondrial genome by RFLP or TTGE analyses. However these approaches require large amounts of DNA or are labor intensive. The use of polymerase amplification of long DNA products (LRPCR) has been described by several groups and more recently summarized by Van Houten's group. The underlying basis use of DNA polymerases capable of generating long DNA products and the rationale is that any lesion (strand breaks, base modifications, apurinic sites) will stop a thermostable DNA polymerase. In this method, band density of the PCR product is quantified either by Southern blotting or binding of a fluorescent dye. Although the latter approach still has some limited use in the study gene expression, it is semi-quantitative and realtime PCR analysis has largely supplanted it. Direct application of real-time PCR to LRPCR has been made difficult because of low processivity and polymerization rates of the DNA polymerases used and SYBR green inhibition of DNA amplification. We have modified the LRPCR protocol to use the commercially available PfuUltra() II Fusion HS DNA Polymerase for real-time determination of mitochondrial DNA amplification as a means to simplify and improve of the accuracy for quantification of mtDNA damage.

The identification of genetically modified (GM) organisms (GMOs) in unknown samples by polymerase chain reaction (PCR) requires the use of a positive control sample, containing the target sequence derived from the respective GMO. For this purpose, either DNA extracted from suitable reference material or plasmids bearing the sequence are used. In the case of isolated genomic DNA, the preparation is cost-intensive and time-consuming, and material availability may be limited. Once the sequence is cloned into a plasmid, it may be simpler and less cost-intensive to purify the DNA, but contamination risk is substantially higher. The potential of multiple displacement amplification (MDA) as a new tool to generate reference material for GMO detection was studied taking the GM maize MON810 as a case. MDA yield of maize-specific DNA and amplification efficiency in dependence of the amount of starting material were estimated using real-time PCR. Applicability of the amplified DNA for the use as reference material was tested with regard to real-time PCR performance and MDA bias. Depending on the amount of genomic DNA (gDNA) input into MDA, amplification rates in the range of 30-fold (100 ng input of gDNA) to 23,000-fold (0.1 ng input of gDNA) were achieved. Real-time PCR performance and gene representation of amplified DNA (mdaDNA) are comparable to those of gDNA. Our results demonstrate that the DNA amplified by MDA is suitable for the use as reference material for qualitative GMO analysis.

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1- previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/microl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/microl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification.

Nucleic acids, especially DNA, are targets of qualitative and quantitative diagnostics for genetically modified organisms (GMO) in seeds, food- and feedstuff. The amplification of the nucleic acid is an essential step for further analyses of the target sequence. The PCR has been the method of choice for DNA amplification in most laboratories, and its real-time version (qPCR) also enables quantitative analysis of target contents. Despite its numerous advantages, PCR technology has some limitations such as the lack of true multiplexing properties. To alleviate the drawbacks linked to PCR technology, alternative nucleic acid amplification methods with promising characteristics are being developed fast. These methods, their advantages, and the inconveniences, which are not yet resolved are summarized in the paper. Special focus is given to the possibilities of using these alternative methods for GMO detection in future, when expansion of GMOs both in diversity and frequencies will make current GMO detection systems difficult to operate.