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BioTechniques Mar 2021This study aimed to develop a quantitative PCR assay to simultaneously quantify human and dog DNA in a multispecies mixture to inform downstream analyses in routine...
This study aimed to develop a quantitative PCR assay to simultaneously quantify human and dog DNA in a multispecies mixture to inform downstream analyses in routine forensic casework and scientific research. The human target is the Yb8 element, which has approximately 2000 copies per cell, and the canine target is the SINEC_Cf element, which has approximately 200,000 copies per cell. The internal positive control is a universal exogenous assay consisting of forward and reverse primers, a NED-labeled probe with an MGBNFQ quencher and a 65-bp synthetic template. Results suggest a potentially robust assay with a fast run time and a high degree of sensitivity, precision and species specificity, with direct application to domestic pet samples in veterinary genetics and forensics.
Topics: Animals; DNA; DNA Primers; Dogs; Forensic Medicine; Humans; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity
PubMed: 33307787
DOI: 10.2144/btn-2020-0144 -
Genomics Sep 2021As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver...
As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.
Topics: Conserved Sequence; DNA Primers; Genome, Viral; Mutation; SARS-CoV-2; Sequence Analysis, DNA; Software
PubMed: 34293476
DOI: 10.1016/j.ygeno.2021.07.020 -
BMC Genomics Jun 2024Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied...
BACKGROUND
Reverse transcription quantitative PCR (RT-qPCR) with intercalating dyes is one of the main techniques to assess gene expression levels used in basic and applied research as well as in diagnostics. However, primer design for RT-qPCR can be complex due to the high demands on primer quality. Primers are best placed on exon junctions, should avoid polymorphic regions, be specific to the target transcripts and also prevent genomic amplification accurately, among others. Current software tools manage to meet all the necessary criteria only insufficiently. Here, we present ExonSurfer, a novel, user-friendly web-tool for qPCR primer design.
RESULTS
ExonSurfer combines the different steps of the primer design process, encompassing target selection, specificity and self-complementarity assessment, and the avoidance of issues arising from polymorphisms. Amplification of potentially contaminating genomic DNA is avoided by designing primers on exon-exon junctions, moreover, a genomic alignment is performed to filter the primers accordingly and inform the user of any predicted interaction. In order to test the whole performance of the application, we designed primer pairs for 26 targets and checked both primer efficiency, amplicon melting temperature and length and confirmed the targeted amplicon by Sanger sequencing. Most of the tested primers accurately and selectively amplified the corresponding targets.
CONCLUSION
ExonSurfer offers a comprehensive end-to-end primer design, guaranteeing transcript-specific amplification. The user interface is intuitive, providing essential specificity and amplicon details. The tool can also be used by command line and the source code is available. Overall, we expect ExonSurfer to facilitate RT-qPCR set-up for researchers in many fields.
Topics: Exons; Software; DNA Primers; Internet; Humans; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 38867172
DOI: 10.1186/s12864-024-10456-2 -
Nature Communications Feb 2021Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of...
Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.
Topics: Amino Alcohols; Base Pairing; Biocatalysis; Butylene Glycols; Cations, Divalent; DNA; DNA Primers; Imidazoles; Manganese; Nucleic Acid Conformation; Nucleic Acids; RNA; Solutions
PubMed: 33547322
DOI: 10.1038/s41467-021-21128-0 -
Scientific Reports Mar 2017Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA...
Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user's input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.
Topics: DNA Primers; Polymerase Chain Reaction; Software; Web Browser
PubMed: 28358011
DOI: 10.1038/srep45349 -
Open Veterinary Journal 2021The rufous-winged buzzard () belongs to the order Accipitriformes, which is monomorphic, resulting in the difficulty to identify the gender. However, sex determination...
BACKGROUND
The rufous-winged buzzard () belongs to the order Accipitriformes, which is monomorphic, resulting in the difficulty to identify the gender. However, sex determination is important for predicting the sex ratio of this buzzard in nature in order to avoid its extinction.
AIM
We aimed to develop a primer set that is able to sex the rufous-winged buzzard through polymerase chain reaction (PCR) amplification and compare the efficacy of the two sets of primers by using PCR technique.
METHODS
In the following, sensitivity refers to the smallest DNA concentration that allowed us to accurately sex a bird and specificity refers to the ability to clearly distinguish the sex based on the visual appearance of the bands. Blood samples were collected from captive buzzards. The DNA was extracted from them and was diluted to 50, 25, 10, 5, 2.5, 1.67, and 1 ng/μl. Two sets of primers, including P2/NP/MP and 2550F/2718R, were used to amplify the chromo-helicase DNA binding (CHD) gene of known gender buzzards using the PCR process to determine gender and to compare their sensitivity. To measure specificity, both primers were used to amplify CHD gene fragment of other unknown gender birds.
RESULTS
The lowest concentration of the DNA template where P2/NP/MP could amplify DNA fragments was 1 ng/μl, and this set of primers could identify the gender of all birds correctly, giving 100% specificity. On the other hand, the 2550F/2718R could amplify the DNA fragments from 5 ng/μl, and it had only 78% specificity.
CONCLUSION
The P2/NP/MP primer set was able to correctly identify the gender of rufous-winged buzzard through PCR amplification with high specificity and sensitivity.
Topics: Animals; Birds; DNA; DNA Primers; Polymerase Chain Reaction; Sex Determination Analysis
PubMed: 35070852
DOI: 10.5455/OVJ.2021.v11.i4.7 -
Molecular Aspects of Medicine 2006The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility,... (Review)
Review
The employment of polymerase chain reaction (PCR) techniques for virus detection and quantification offers the advantages of high sensitivity and reproducibility, combined with an extremely broad dynamic range. A number of qualitative and quantitative PCR virus assays have been described, but commercial PCR kits are available for quantitative analysis of a limited number of clinically important viruses only. In addition to permitting the assessment of viral load at a given time point, quantitative PCR tests offer the possibility of determining the dynamics of virus proliferation, monitoring of the response to treatment and, in viruses displaying persistence in defined cell types, distinction between latent and active infection. Moreover, from a technical point of view, the employment of sequential quantitative PCR assays in virus monitoring helps identifying false positive results caused by inadvertent contamination of samples with traces of viral nucleic acids or PCR products. In this review, we provide a survey of the current state-of-the-art in the application of the real-time PCR technology to virus analysis. Advantages and limitations of the RQ-PCR methodology, and quality control issues related to standardization and validation of diagnostic assays are discussed.
Topics: Computer Systems; DNA Primers; DNA Probes; Forecasting; Humans; Molecular Diagnostic Techniques; Polymerase Chain Reaction; Quality Control; Virus Diseases
PubMed: 16481036
DOI: 10.1016/j.mam.2005.12.001 -
BMC Biotechnology Mar 2022Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have...
BACKGROUND
Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired.
METHOD
We present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures).
RESULTS
We first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences.
CONCLUSION
We employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).
Topics: DNA Primers; Mutagenesis; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Sepharose
PubMed: 35282829
DOI: 10.1186/s12896-022-00740-y -
PCR Methods and Applications Apr 1995
Topics: Alkaline Phosphatase; Base Sequence; DNA; DNA Ligases; DNA Primers; DNA, Complementary; Humans; Indicators and Reagents; Molecular Sequence Data; Nucleic Acid Conformation; Phosphorylation; Polymerase Chain Reaction; Restriction Mapping
PubMed: 7580908
DOI: 10.1101/gr.4.5.s195 -
Nucleic Acids Research Jul 2019Quality 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 (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.
Topics: Algorithms; Base Pair Mismatch; Binding Sites; DNA Primers; Genome, Human; Humans; Multiplex Polymerase Chain Reaction; Polymerase Chain Reaction; Quality Control; Sequence Analysis; Software
PubMed: 31066442
DOI: 10.1093/nar/gkz351