Design Polymerase Chain Reaction Primers Research Articles

Computer Program for Primer Design for Loop-Mediated Isothermal Amplification (LAMP)

Introduction. To date, numerous methods of nucleic acid amplification have been proposed, and each method has a number of advantages and disadvantages. One of the most popular methods is Loop-mediated isothermal AMPlification (LAMP). Unlike thermocyclic reactions, such as PCR (polymerase chain reaction), which require three temperature changes and expensive equipment, in LAMP, the entire reaction takes place at one and the same temperature and at the maximum rate possible. An important component of LAMP-amplification is primers (usually 20–25 nucleotides), which need to be matched to a specific part of the nucleotide sequence. It is known that DNA sequence contains four nucleotides: A — adenine and T — thymine, G — guanine and C — cytosine. There is a huge variety of permutations of these nucleotides, and it is practically impossible to analyze such a large amount of data manually. Therefore, there is a need to use modern computer technologies. More than 150 computer programs have been proposed for the design of PCR primers, while for LAMP-primers there are less than 10 of them, and each of them has a number of drawbacks, e.g., in terms of the length of the analyzed site. Therefore, this work is aimed at developing a new domestic computer program for the design of specific primers for LAMP.Materials and Methods. The primer search algorithm was based on a linear search for a substring in a string, taking into account the criteria of primer selection for LAMP. The program complex of LAMP-primer design was implemented in Python programming language. The bioPython library was used to work with various DNA and RNA, and the Qt framework was used to develop the interface.Results. A modification of the direct sampling method using a stencil approach was proposed, taking into account the GC composition and annealing temperature of primers depending on their structure. A software package with a friendly interface was developed. It took into account the design criteria of primers: certificates of registration of computer programs (LAMPrimers iQ No. 2022617417 dated April 20, 2022, LAMPrimers iQ_loop No. 2023662840 dated June 14, 2023) were received. The program is in the public domain at https://github.com/Restily/LAMPrimers-iQDiscussion and Conclusion. The developed software packages can be used for research and analysis in molecular biology and genetics, to create diagnostic test systems that provide high sensitivity and reliability of detection of specific DNA and RNA. The software packages can be used in research institutes and laboratories engaged in the amplification of nucleic acids. The results of evaluating the selected sets of primers for the LAMP reaction were tested, and the effectiveness of working sets using the LAMPrimers iQ program was experimentally proven by the example of the detection of genetic material of the SARS-CoV-2 coronavirus.

DeGenPrime provides robust primer design and optimization unlocking the biosphere.

Polymerase chain reaction (PCR) is the world's most important molecular diagnostic with applications ranging from medicine to ecology. PCR can fail because of poor primer design. The nearest-neighbor thermodynamic properties, picking conserved regions, and filtration via penalty of oligonucleotides form the basis for good primer design. DeGenPrime is a console-based high-quality PCR primer design tool that can utilize MSA formats and degenerate bases expanding the target range for a single primer set. Our software utilizes thermodynamic properties, filtration metrics, penalty scoring, and conserved region finding of any proposed primer. It has degeneracy, repeated k-mers, relative GC content, and temperature range filters. Minimal penalty scoring is included according to secondary structure self-dimerization metrics, GC clamping, tri- and tetra-loop hairpins, and internal repetition. We compared PrimerDesign-M, DegePrime, ConsensusPrimer, and DeGenPrime on acceptable primer yield. PrimerDesign-M, DegePrime, and ConsensusPrimer provided 0%, 11%, and 17% yield, respectively, for the alternative iron nitrogenase (anfD) gene target. DeGenPrime successfully identified quality primers within the conserved regions of the T4-like phage major capsid protein (g23), conserved regions of molybdenum-based nitrogenase (nif), and its alternatives vanadium (vnf) and iron (anf) nitrogenase. DeGenPrime provides a universal and scalable primer design tool for the entire tree of life. DeGenPrime is written in C++ and distributed under a BSD-3-Clause license. The source code for DeGenPrime is freely available on www.github.com/raw-lab/degenprime.

Evaluating the Usefulness of a Large Language Model as a Wholesome Tool for De Novo Polymerase Chain Reaction (PCR) Primer Design.

This study aimed to assess the ability of language learning models (LLMs), specifically GPT-3.5 (Chat Generative Pre-trained Transformer 3.5) and GPT-4 (Chat Generative Pre-trained Transformer 3.5), in designing primers for diagnostic polymerase chain reaction (PCR) of the monkeypox virus (MPXV). Five primer pairs were generated by each LLM, and their thermodynamic properties and specificity were analysed post-hoc using commonly used software. The LLMs demonstrated ability in sequence generation and predicting melting temperatures (Tm), but their accuracy in predicting GC content was suboptimal, necessitating further investigation. Results indicated that, of the total primer pairs, only three designed by GPT-4 and two by GPT-3.5 could theoretically form a PCR product, but only one pair demonstrated suitable parameters for experimental validation. This preliminary exploration suggests that while LLMs have a potential in aiding primer design, their accuracy needs improvement to match current deterministic, rule-based tools used in the field. Consequently, manual intervention remains a crucial step in PCR primer design.

Molecular effects of site-specific phosphate-methylated primer on the structure and motions of Taq DNA polymerase

Polymerase chain reaction (PCR) is a powerful molecular biology assay for gene detection and quantification. Conventional DNA primers for PCR often suffer from poor sensitivity in specific gene detection. Recently, oligonucleotides containing methyl phosphotriester (MPTE-DNA) have been developed with enhanced DNA hybridization and improved gene detection sensitivity. Yet, site-specific MPTE-modifications on DNA primers have been reported to affect PCR amplification efficiencies while the detailed mechanism remains elusive. Here, we utilized molecular dynamics (MD) simulation to examine the effects of site-specific MPTE-modified primers on the structure and motions of DNA/Taq polymerase complexes. All tested MPTE-DNA/Taq complexes exhibited RMSD values below 2 Å, indicating insignificant effects of all methylation sites on the complex stability. The energetic and hydrogen-bonding analyses suggest minor effects of methylation at t-3, t-4, t-6, and t-7 positions on the DNA−Taq interaction. Principal component analyses further reveal that only t-3, and t-7 methylations preserve the motions of the Taq thumb domain. The site-specific methylation affects the interaction between DNA and the surrounding protein residues, resulting in allosteric-like effects on the DNA/Taq complex. The MD data complement the best experimentally observed PCR efficacies for the t-3 and t-7 positions among all tested MPTE-primers. The unveiled molecular insights can benefit the design of novel PCR primers for improving genetic testing platforms.

Multiobjective optimization-driven primer design mechanism: towards user-specified parameters of PCR primer.

Primers are critical for polymerase chain reaction (PCR) and influence PCR experimental outcomes. Designing numerous combinations of forward and reverse primers involves various primer constraints, posing a computational challenge. Most PCR primer design methods limit parameters because the available algorithms use general fitness functions. This study designed new fitness functions based on user-specified parameters and used the functions in a primer design approach based on the multiobjective particle swarm optimization (MOPSO) algorithm to address the challenge of primer design with user-specified parameters. Multicriteria evaluation was conducted simultaneously based on primer constraints. The fitness functions were evaluated using 7425 DNA sequences and compared with a predominant primer design approach based on optimization algorithms. Each DNA sequence was run 100 times to calculate the difference between the user-specified parameters and primer constraint values. The algorithms based on fitness functions with user-specified parameters outperformed the algorithms based on general fitness functions for 11 primer constraints. Moreover, MOPSO exhibited superior implementation in all experiments. Practical gel electrophoresis was conducted to verify the PCR experiments and established that MOPSO effectively designs primers based on user-specified parameters.

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Sanger Sequencing for Molecular Diagnosis of SARS-CoV-2 Omicron Subvariants and Its Challenges

Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The European Centre for Disease Prevention and Control (eCDC) and the World Health Organization (WHO) recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the polymerase chain reaction (PCR)-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490 - 509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariants when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.2 RBD and an atypical BA.1 NTD, which can only be detected by using specially designed PCR primers. In addition, Sanger sequencing may reveal unclassified subvariants, such as BA.4/BA.5 with L84I mutation in the S gene NTD. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.

Development of Multiplex PCR Coupled DNA Chip Technology for Assessment of Endogenous and Exogenous Allergens in GM Soybean.

Allergenicity assessment of transgenic plants and foods is important for food safety, labeling regulations, and health protection. The aim of this study was to develop an effective multi-allergen diagnostic approach for transgenic soybean assessment. For this purpose, multiplex polymerase chain reaction (PCR) coupled with DNA chip technology was employed. The study was focused on the herbicide-resistant Roundup Ready soya (RRS) using a set of certified reference materials consisting of 0, 0.1%, 0.5%, and 10% RRS. Technically, the procedure included design of PCR primers and probes; genomic DNA extraction; development of uniplex and multiplex PCR systems; DNA analysis by agarose gel electrophoresis; microarray development, hybridization, and scanning. The use of the asymmetric multiplex PCR method is shown to be very efficient for DNA hybridization with biochip probes. We demonstrate that newly developed fourplex PCR methods coupled with DNA-biochips enable simultaneous identification of three major endogenous allergens, namely, Gly m Bd 28K, Gly m Bd 30K, and lectin, as well as exogenous 5-enolppyruvyl shikimate-phosphate synthase (epsps) expressed in herbicide-resistant roundup ready GMOs. The approach developed in this study can be used for accurate, cheap, and fast testing of food allergens.

The Significance of PCR Primer Design in Genetic Diversity Studies: Exemplified by Recent Research into the Genetic Structure of Marine Species.

Genetic markers are widely applied in the study of genetic diversity for many species. The approach incorporates a Polymerase Chain Reaction (PCR) amplification of targeted sequences in the genome. Crucial for the overall success of a PCR experiment is the careful design of synthetic oligonucleotide primers. Ideally designed primer pairs will ensure the efficiency and specificity of the amplification reaction, resulting in a high yield of the desired amplicon. Important criteria such as primer-sequence, -length, and -melting temperature (Tm) are fundamental for the selection of primers and amplification of targeted nucleotide sequences from a DNA template. There are many computational tools available to assist with critical bioinformatics issues related to primer design. These resources allow the user to define parameters and criteria that need to be taken into account when designing primers. Following the initial in silico selection, a primer pair should be further tested in vivo for their amplification efficiency and robustness.Using examples taken from genetic diversity studies in a marine crustacean, this chapter provides outlines for the application of PCR technology and discusses details for the design of primers for the development and characterization of microsatellite and SNP-markers.

Degenerate PCR primers for assays to track steps of nitrogen metabolism by taxonomically diverse microorganisms in a variety of environments

Steps in the global nitrogen cycle are mainly catalyzed by microorganisms. Accordingly, the activities of these microorganisms affect the health and productivity of ecosystems. Their activities are also used in wastewater treatment systems to remove reactive nitrogen compounds and prevent eutrophication events triggered by nutrient discharges. Therefore, tracking the activities of these microorganisms can provide insights into the functioning of these systems. The presence and abundance of genes encoding nitrogen-metabolizing enzymes can be traced via polymerase chain reaction (PCR); however, this requires primers that are sensitive to a heterogenous gene pool yet specific enough to the target biomarker. The ever-expanding diversity of sequences available from databases includes many sequences relevant to nitrogen metabolism that match poorly with primers previously designed to track their presence and/or abundance. This includes genes encoding ammonia monooxygenase (AMO) of ammonia oxidizing microorganisms, nitrite oxidoreductase (NXR) of nitrite oxidizing bacteria, and nitrous oxide reductase (NOS) of denitrifying bacteria. Some primers are also not designed to generate the short (~200 nucleotides) amplicons required for real-time quantitative PCR (qPCR) and reverse-transcriptase qPCR (qRT-PCR). In this study, genes collected from the Integrated Microbial Genomes database (IMG) were aligned to design PCR primers that could capture more sequence diversity than is possible using existing primers. Primers were designed to target three clades of AMO (Betaproteobacteria, Chrenarchaeota, and complete ammonia oxidizing Nitrospira), periplasmic NXR and two clades of NOS (Proteobacteria and Bacteroidetes/Firmicutes). These primers successfully amplified target sequences from two wastewater treatment plants with biological nitrogen removal (one with simultaneous nitrification/denitrification and one with distinct anoxic/oxic zones) and estuary sediment. Nucleotide sequences of the amplicons retrieved homologs when used to query GenBank by BLAST. While convincingly identified as target sequences for these primer pairs, these amplicons were divergent from each other, and quite divergent (as low as 73%) from those present in GenBank, suggesting these primers are capable of capturing a diverse range of sequences. A direct comparison showed that primers designed here are better suited to environmental samples, such as wastewater treatment facilities, by producing a greater number of amplicons from the same sample than primers currently established in literature.

MFEprimer-3.0: quality control for PCR primers.

Quality control (QC) for lab-designed primers is crucial for the success of a polymerase chain reaction (PCR). Here, we present MFEprimer-3.0, a functional primer quality control program for checking non-specific amplicons, dimers, hairpins and other parameters. The new features of the current version include: (i) more sensitive binding site search using the updated k-mer algorithm that allows mismatches within the k-mer, except for the first base at the 3′ end. The binding sites of each primer with a stable 3′ end are listed in the output; (ii) new algorithms for rapidly identifying self-dimers, cross-dimers and hairpins; (iii) the command-line version, which has an added option of JSON output to enhance the versatility of MFEprimer by acting as a QC step in the ‘primer design → quality control → redesign’ pipeline; (iv) a function for checking whether the binding sites contain single nucleotide polymorphisms (SNPs), which will affect the consistency of binding efficiency among different samples. In summary, MFEprimer-3.0 is updated with the well-tested PCR primer QC program and it can be integrated into various PCR primer design applications as a QC module. The MFEprimer-3.0 server is freely accessible without any login requirement at: https://mfeprimer3.igenetech.com/ and https://www.mfeprimer.com/. The source code for the command-line version is available upon request.

Power of PCR pre‐labs and a co‐mentoring community group: Increasing impacts on skills and confidence

Science concept recall and engagement take place through individual pre‐lab preparation, in‐lab partnerships, hands‐on training, and social learning communities within the Social Ecological Model and Cognitive Theory frameworks. STEM majors are introduced to key concepts and skills at several stages in pre‐requisite courses required for later enrollment in our Biochemistry Laboratory course. Because this curriculum assumes high recall, we measured student understanding and recall in a series of pre‐skill tests at the lab course onset: Polymerase Chain Reaction (PCR) ability to create potential primers for a strand of DNA; math dilution skills and ability to calculate molarity for a buffer solution; and other concepts assumed to be easily recalled for application to lab course exercises. Per common practice, we employed pre‐lab exercises to help refresh students' memories and ensure lab preparation. A surprising gap in understanding of PCR was detected, so pre‐lab interventions were investigated along with near‐peer mentoring in a co‐mentoring community group to promote learning inclusion.Spanning iterative semesters (Fall 2016–Fall 2018) of an upper‐level Biochemistry lab course, we used pre‐post assessments of de‐identified student data per IRB (sample size n=56–90 per semester) to investigate 1) recall, 2) iterative versions of pre‐lab prompts for enhanced skill and confidence, and 3) different student outcomes per intervention. Skill assessments were qualitatively coded and hand‐scored for correctness, analyzed, and matched to paired pre‐post student self‐report in a 5‐point Likert scale of confidence in theory and confidence in skill: Doing PCR, Designing PCR Primers, Translation of DNA, Dilution Calculations, Molarity Calculations, etc.Student recall on the first lab day varied widely in pre‐assessments. Confidence in the theory and skill trended with higher incoming self‐reported confidence than actual ability to design a primer, and higher confidence with theory than with skill. Results varied among no pre‐lab, paper‐based, and online links with videos suggesting overall increased success in skill and confidence measured in post‐course assessments, yet post‐course student outcomes were still suboptimal. An additional post‐course intervention recruiting students for a co‐mentoring community model with a more course‐based undergraduate research (CUR) approach provided deeper learning out of class, social support, and additional outcomes.Use of pre‐labs was valuable to promote recall; however, pre‐lab effectiveness required deeper prompts. Simply reading and doing PCR in lab was not sufficient, nor was repeated lecture, discussion, or quizzing. The co‐mentoring community model extended the course learning to more applied learning and broader impacts building STEM learning and inclusion.Support or Funding InformationUniversity of Minnesota Grant‐in‐AidThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polymerase Chain Reaction (PCR) Detection of 3C Ligation Products Present in 3C, ChIP-Loop, and Control Libraries: Library Titration and Interaction Frequency Analysis.

In all 3C-based assays, the aim is to measure the frequency with which two loci interact within cells. This is achieved by determining the relative abundance of ligation products formed during the 3C protocol that carry sequences from both loci. Individual 3C ligation products that are present in a 3C library, the ChIP-loop library, or the control library can be detected by polymerase chain reaction (PCR) using locus-specific primers. The following protocol outlines the procedure to measure relative abundances of ligation products in these libraries using PCR. The first part of the protocol-library titration-is required only once to determine the linear range of PCR detection. Once the linear range of PCR detection is known for a given ligation product library, follow the second part-interaction analysis-to measure the abundance of ligation products of interest in that library. Specific issues regarding determining the relative abundance of ligation products, designing PCR primers, and quantifying interaction frequencies are also described.

Primer Server - A web application to design primers for the amplification of unique DNA targets in complex genomes

Polymerase Chain Reaction (PCR) is one of the most important inventions of the 20th century in molecular biology. PCR is a technique to amplify or make in a test tube many copies of a specific DNA region. Miniscule amounts of the genetic material from any organism can now be amplified to identify individuals, manipulate DNA, detect infectious organisms including the viruses that cause AIDS, hepatitis, and tuberculosis, detect genetic variations including mutations in genes, and numerous other tasks. PCR primers are short, single-stranded DNAs that define the section of DNA to be amplified. Two primers are used in each PCR reaction, designed so that they bind at flanking locations surrounding the target region. Critically, off-target binding may lead to experimental failure or worse, to misleading results. Thus, potential primers of approximately 20 DNA bases in length, must be examined for off-target binding among, for example, the 3.2 billion DNA bases from all human chromosomes, the human genome. The purpose of our study is to make a user-friendly tool (Primer Server) that can design PCR primers efficiently and accurately as well as visualize the designed primers. Our web-based bioinformatics tool selects optimal primer sequences within the starting material by using a C module called primer3 and then prioritizing and/or eliminating potential primers based on comparison of the primer bases against all bases in the genome using an algorithm called BLAST. This tool has an easy-to-use interface which was designed using Angular2, and an efficient server-side code written in Python. While similar tools exist, our tool is more user-friendly, efficient and uses extensive form validation to minimize errors in the user input. Our tool can be used to design primers that will be used in laboratory experiments to amplify DNA from various organisms, including large, complex genomes such as humans, other animals and plants.

RANCANGAN PRIMER SPESIFIK GEN MACROPHAGE MANNOSE RECEPTOR (MMR) UNTUK POLYMERASE CHAIN REACTION (PCR) DAN SEKUENSING DEOXYRIBO NUCLEIC ACID (DNA)

The order (sequencing) determinationof DeoxyribonucleicAcid (DNA) bases is the gene's most basic information, using the methodof Polymerase Chain Reaction (PCR) as its stage. A key factor of successful detection by PCR is specific PCR primer design choice. Thedetection of diversity of Mycobacterium Mannose Receptor (MMR) gene, responsible for recognizing mannosylated antigen structureof Mycobacterium tuberculosis (M.tb) by DNA sequencing of exon 7 chromosome 10p12, related to susceptiblity for PulmonaryTuberculosis(TB), was first performed in China in 2012. The purpose of this study was to find specific primerfromboth design originatedfrom the research in China/primer I and my own design/primer IIby using Primer3 software. This study was based on 10 healthy subjectsand was a preliminary study of a research titled. The Relationship of Single Nucleotide Polymorphisms (SNPs) of Macrophage MannoseReceptor Gene to Pulmonary Tuberculosis Cases. The examination materials consist of 3 mL of EDTA blood and DNA extraction from itsbuffy coat. The resulting DNA was processed by PCR to amplify MMR gene with primer I and II. The primer I successfully amplified DNAfragments up to 780bp while primer II only 329 bp. The MMR gene DNA sequencing analysis was performed on the amplification resultof both kinds primers by using DNA Baser and Ensembl−BLAST software. The results were different, DNA sequencing result by using theprimer I was found in several chromosomes and also in several loci. Whereas, by using the primer II, it was only found in chromosome10 and in the same locus. Based on this study, it can be concluded that the specific primer design is one of the most important factorsin the success of DNA sequencing.

IndCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

CEMAsuite: open source degenerate PCR primer design.

The codon-equivalent multiple alignment suite begins conservational analysis for polymerase chain reaction primer design at the protein level, allowing the user to design consensus primers capable of detecting homologous coding sequences even when low-to-moderate sequence information is available. This package also condenses the wealth of information associated with multiple sequence alignments and presents them in an intuitive manner, allowing the user to quickly and effectively address degenerate primer design considerations. https://sourceforge.net/projects/cemasuite/. benton@lsu.edu or cemasuite@gmail.com Supplementary data are available at Bioinformatics online.

Highly efficient identification of thousands of microsatellite loci in the pearl oyster (Pinctada martensii) from RNA-Seq

High-throughput RNA-Seq affords a cost and time effective means of obtaining large numbers of genetic markers for aquatic genomics. Here, we present thousands of novel microsatellite loci developed for the pearl oyster, Pinctada martensii from the Illumina HiSeq™ 2000 library of the pearl sac. Free user-friendly bioinformatics tools were employed to screen for microsatellite loci and design appropriate primers in 102,762 unigenes with 7216 microsatellite loci identified in total, 4862 of which had flanking sequences suitable for polymerase chain reaction primer design. The 50 randomly chosen primer pairs were tested in two populations of pearl oyster (base population (POP1) and selected population (POP2), with 30 individuals of each population). All the primer pairs were amplified successfully in two populations. All loci were polymorphic in POP1, while there were 3 loci showing monomorphism in POP2. In POP1 and POP2, observed heterozygosity from 0.033 to 1.000 and 0.000 to 1.000, 19 and 16 microsatellite loci deviated significantly from Hardy–Weinberg expectations including a Bonferroni correction (P < 0.001). Thirteen loci were highly informative content (PIC ≥ 0.5) in both populations. These identified loci will be useful for potential application for evolutionary, population genetic and chromosome linkage mapping research on pearl oyster.

Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.

BackgroundOver the last decades, molecular cloning has transformed biological sciences. Having profoundly impacted various areas such as basic science, clinical, pharmaceutical, and environmental fields, the use of recombinant DNA has successfully started to enter the field of cellular engineering. Here, the polymerase chain reaction (PCR) represents one of the most essential tools. Due to the emergence of novel and efficient PCR reagents, cloning kits, and software, there is a need for a concise and comprehensive protocol that explains all steps of PCR cloning starting from the primer design, performing PCR, sequencing PCR products, analysis of the sequencing data, and finally the assessment of gene expression. It is the aim of this methodology paper to provide a comprehensive protocol with a viable example for applying PCR in gene cloning.ResultsExemplarily the sequence of the tdTomato fluorescent gene was amplified with PCR primers wherein proper restriction enzyme sites were embedded. Practical criteria for the selection of restriction enzymes and the design of PCR primers are explained. Efficient cloning of PCR products into a plasmid for sequencing and free web-based software for the consecutive analysis of sequencing data is introduced. Finally, confirmation of successful cloning is explained using a fluorescent gene of interest and murine target cells.ConclusionsUsing a practical example, comprehensive PCR-based protocol with important tips was introduced. This methodology paper can serve as a roadmap for researchers who want to quickly exploit the power of PCR-cloning but have their main focus on functional in vitro and in vivo aspects of cellular engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-1611-9-2) contains supplementary material, which is available to authorized users.

Primer design for PCR reactions in forensic biology.

The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.