Webtools for PCR, qPCR, in silico PCR and oligonucleotides

PCR primer design, in silico PCR, oligonucleotide assembly and analyses

PCR tool provides comprehensive and professional facilities for designing primers for most PCR applications and their combinations: standard, multiplex, long distance, inverse, real-time, Xtreme Chain Reaction (XCR®), group-specific (universal primers for phylogenetically related DNA sequences) and unique (specific primers for each from phylogenetically related DNA sequences), bisulphite modification assays, polymerase extension PCR multi-fragments assembly cloning (OE-PCR) and Loop-mediated Isothermal Amplification (LAMP); microarray design; Allele-Specific PCR (AS-PCR) - KASP™ (Kompetitive Allele Specific PCR) or PACE™ (PCR Allele Competitive Extension) based genotyping assay design for biallelic discrimination of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci;
in silico (virtual) PCR or primers and probes search or in silico PCR against whole genome(s) or a list of chromosome - prediction of probable PCR products and search of potential mismatching location of the specified primers or probes;
comprehensive primer test, the melting temperature calculation for standard and degenerate oligonucleotides including LNA and other modifications, primer PCR efficiency, primer's linguistic complexity, and dilution and resuspension calculator;
analyzes different features of multiple primers simultaneously, the melting temperature, GC content, sequence linguistic complexity, primer PCR efficiency and molecular weight, the extiction coefficient, the optical density (OD);
primers (probes) are analyzed for all primer secondary structures including the alternative hydrogen bonding to Watson-Crick base pairing such as G-quadruplexes or wobble base pairs (like G-G, G-T, G-A), hairpins, self-dimers and cross-dimers in primer pairs;
tool calculate Tm for primer dimers with mismatches for pure and mixed bases using averaged nearest neighbour thermodynamic parameters and for modifications (inosine, uridine, or LNA);
tool for identifying simple sequence repeat (SSR) loci by analysing the low complexity regions of input sequences;
tool for restriction I-II-III types enzymes analysis, find or create restriction enzyme recognition sites for coding sequences;
tool for searching for similar sequences (or primers);
translates nucleotide (DNA/RNA) sequences to the corresponding peptide sequence in all six frames for standard and degenerate DNA and modifications (inosine, uridine);
Polymerase Chain Assembly (PCA) - created to automate the design oligonucleotide sets for long sequence assembly by PCR;
the program includes various bioinformatics tools for patterns analysis of sequences with GC:(G-C)/(G+C), AT:(A-T)/(A+T), SW:(S-W)/(S+W), MK:(M-K)/(M+K), purine-pyrimidine (R-Y)/(R+Y) skews, CG% and GA% content and the melting temperature and considers linguistic sequence complexity profiles.

jPCR Web Start


provides comprehensive analysis of sequence with standard and mixed bases, as well as DNA, RNA, methylated, locked and phosphorothioated bases; tool will calculate the physical properties of the sequence including length, CG content, melting temperature, molecular weight, the extiction coefficient, the optical density (OD), sequence linguistic complexity, primer PCR efficiency, self-dimers and G-quadruplexes detection.
The melting temperature calculations are based on nearest neighbour thermodynamic parameters for standard and degenerate oligonucleotides including LNA and other modifications. And provides a dilution and resuspension calculator for stocks.

PrimerAnalyser Web Start


Analyzes different features of multiple primers simultaneously, the melting temperature calculation for standard and degenerate oligonucleotides, GC content, primer PCR efficiency, sequence linguistic complexity, molecular weight, the extiction coefficient, the optical density (OD);
primers are analyzed for all primer secondary structures including G-quadruplexes detection, hairpins, self-dimers and cross-dimers in primer pairs;
the optimal temperature of annealing (Ta) calculation for all given primers.

PrimersList Web Start

PCR reaction setup calculators

PCR, qPCR and LAMP reaction setup calculator; tool for planning PCR, qPCR and LAMP (Loop-mediated Isothermal Amplification) reactions, mixing solutions.

PCR reaction setup calculators Web Start

Universal dilution and mixing two solutions calculator

Dilution calculator, applicable for mixing two solutions with various concentrations (molar or %, or other), as well as for various pH or mixing stock solution with solvent like water.

jPCR Web Start

Comparison of primer design and oligonucleotide analysing tools versus other most popular on-line primer design and analysing packages.

The tools are written in Java with NetBeans IDE (Apache) and require the Java 8 Runtime Environment (Java SE 8 Platform) or use OpenWebStart an open source reimplementation of the Java Web Start technology for latest Java SE 11 (LTS) or Java SE 16.

Understanding a Java 8 error message:
If the Java security level is set to "Very High" then no unsigned Java programs are allowed to execute.

Add http://primerdigital.com/ site to "Exception Site List", and set "Security Level" to High.
Download a Certificate file, import it to "Signer CA" at Java Control Panel:
Java Control Panel

Dr. Ruslan Kalendar

Reference apply to all Web Tools update, if you use it in your work please cite:

Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI 2017. FastPCR: an in silico tool for fast primer and probe design and advanced sequence analysis. Genomics, 109: 312-319. DOI:10.1016/j.ygeno.2017.05.005

Kalendar R, Muterko A, Shamekova M, Zhambakin K 2017. In silico PCR tools a fast primer, probe and advanced searching. Methods in Molecular Biology, 1620: 1-31. DOI:10.1007/978-1-4939-7060-5_1

Kalendar R, Tselykh T, Khassenov B, Ramanculov EM 2017. Introduction on using the FastPCR software and the related Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Methods in Molecular Biology, 1620: 33-64. DOI:10.1007/978-1-4939-7060-5_2


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