Webtools for PCR, qPCR, in silico PCR and oligonucleotides
PCR tool provides comprehensive and professional facilities for designing primers for most PCR applications and their combinations: standard, multiplex, long distance, inverse, real-time, 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 microarray design;
(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 and homing endonucleases 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) or oligos assembly - created to automate the design oligonucleotide sets for long sequence assembly by ligase chain reaction (LCR) and 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% content and the melting temperature and considers linguistic sequence complexity profiles.
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.
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.
PCR and qPCR reaction setup calculator; tool for planning PCR and qPCR reactions, mixing solutions.
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.
The tools are written in Java with NetBeans IDE
) and require the Java Runtime Environment (JRE
) on a computer.
To determine which version of Java your web browser is using, please visit javatester or java.com.
UNDERSTANDING A JAVA ERROR MESSAGE:
If the Java 7 security level is set to "Very High" then no unsigned Java programs are allowed to execute. The error message is "Application Blocked by Security Settings ... Your security settings have blocked an untrusted application from running". The message refers to an "untrusted" application when it really is describing an unsigned application.
set "Security Level" to Medium (Least secure setting - All Java applications will be allowed to run after presenting a security prompt) and add http://primerdigital.com/ site to "Exception Site List":
Optionally: Install Java 8 (No Windows XP support on Java 8).
Reference apply to all Web Tools update, if you use it in your work please cite:
Kalendar R, Lee D, Schulman AH 2011. Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics, 98(2): 137-144. DOI: 10.1016/j.ygeno.2011.04.009
[Most Cited Genomics Articles] [Most Downloaded Genomics Articles]
Kalendar R, Lee D, Schulman AH 2014. FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. DNA Cloning and Assembly Methods, Methods in Molecular Biology, Svein Valla and Rahmi Lale (ed.), Humana Press, 1116: 271-302. ISBN 978-1-62703-763-1, DOI: 10.1007/978-1-62703-764-8_18
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