Comprehensive Web Tools for Advanced PCR Design, Quantitative Fluorescent PCR (QF-PCR), Genotyping, Loop-mediated Isothermal Amplification (LAMP), Virtual PCR, Gibson Assembly, Multiplex Tiling PCR panel design, Oligonucleotide Analysis, and Repeat Sequence Identification
by Ruslan Kalendar Email: [email protected]
If you use or modify this code in your work, please include a citation and a link to the original repository:
Based on the work by Ruslan Kalendar – (https://github.com/rkalendar/)
Kalendar R. 2025. Comprehensive web-based platform for advanced PCR design, genotyping, synthetic biology, molecular diagnostics, and sequence analysis. Molecular Therapy Nucleic Acids, 36(4): 102716. DOI:10.1016/j.omtn.2025.102716
https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(25)00270-7
https://www.sciencedirect.com/science/article/pii/S2162253125002707
Your acknowledgment is greatly appreciated!
https://primerdigital.com/tools/
Programming language: JavaScript Other requirements: Modern web browser (e.g., Chrome, Firefox, Edge)
To launch the application use file: /sites/index.html
This application offers advanced capabilities for designing primers across a wide range of PCR applications, including standard, inverse, multiplex, quantitative fluorescence (TaqMan and MGB-probe assay design), and bisulfite PCR. It also supports the development and validation of primer sets for genotyping single nucleotide polymorphisms (SNP) and insertions/deletions (InDel). Additionally, all individual tasks can be efficiently multiplexed for high-throughput analysis, such as fluorescence probe-based multiplex real-time qPCR assays.
Allele-Specific PCR (AS-PCR) - KASP™ (Kompetitive Allele Specific PCR) or PACE™ (PCR Allele Competitive Extension) or Allele-Specific Quantitative PCR (ASQ) - based genotyping assay designs for multiallelic discrimination of single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) at specific loci. The application for genotyping assay design for SNP/InDel-specific KASP assay-targeting primers (KASP Assay Mix).
Loop-mediated Isothermal Amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA for a highly specific amplification reaction. LAMP is a highly sensitive, specific, and rapid DNA amplification technique that has revolutionized molecular biology research and clinical diagnostics. LAMP reactions occur under isothermal conditions, and do not require special thermal cyclers. The LAMP assay can also be used for bisulfite LAMP primer design.
Custom multiplex tiling PCR panel design for amplicon sequencing technology, targeting next-generation (NGS) and third-generation (TGS) DNA sequencing technologies. Multiplex amplicon sequencing is a powerful method suitable for targeted sequencing across a wide range of organisms, including humans, plants, animals, and microorganisms - assuming that reference genome sequences are available. This tool enables users to design custom multiplex tiling PCR panels optimized for high-throughput amplicon sequencing.
Virtual (in silico) or electronic PCR (ePCR) primers/probes or microRNA (miRNA) or a target sequence complementary to the CRISPR RNA (crRNA) on the guide RNA (with a focus on eliminating "off-target" effects) sequences search against whole genome (chromosome) prediction of likely PCR products and search for potential mismatches of the specified primers or probes. Search for multiple targets simultaneously within a specified range. In silico PCR primer searching is useful for the discovery of melting temperature target-binding sites.
Tool for de novo identification, masking, and clustering of all repeated sequences at the genomic scale. This tool detects DNA sequences with interspersed repeats and low-complexity DNA sequences (simple sequence repeats or microsatellites, telomers, and satellite sequences).
Gibson Cloning is a technique for assembling DNA constructs that allows multiple linear segments to be joined either into a single large linear segment or, if the segments contain the appropriate components and overlap, into an intact plasmid. An isothermal, single-reaction method for the assembly of multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
It provides a comprehensive analysis of sequences with both standard and mixed bases. The tool calculates the physical properties of the sequence, including the length, GC content, melting temperature, molecular weight, extinction coefficient, optical density (OD), sequence linguistic complexity, and self-dimer detection. The melting temperature calculations were based on nearest-neighbor thermodynamic parameters for standard and mixed oligonucleotides, including LNA modifications. It also provides a dilution and resuspension calculator for the stocks.
PrimersPrimersList Analyzes different features of multiple primers simultaneously, including the melting temperature calculation for standard and mixed oligonucleotides, GC content, sequence linguistic complexity, molecular weight, the extinction coefficient, and the optical density (OD); primers are analyzed for all secondary structures, including self-dimers and cross-dimers in primer pairs.
PCR, Loop-mediated Isothermal Amplification (LAMP) or any reaction setup calculator.
A dilution calculator is applicable for mixing two solutions with various concentrations (molar, %, or other), different pH levels, or mixing the stock solution with a solvent like water.