● Recommended Citation
Please cite these papers when using FastPCR and Web Tools
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2026
Kalendar R, Daniyarov A, Serikzhan A, Kairov U, Basharova D, Toleuzhanova A, Bekbayeva A, Romanova A, Zholdybayeva PCRpanel: automated design of ultra-multiplex PCR panels for targeted amplicon sequencing. BMC Bioinformatics.
DOI PDF -
2026
Kalendar R. Fast end-to-end design of complete LAMP assays in the browser, from masked template to dimer-screened primer set. Frontiers in Bioinformatics.
DOI PDF -
2025
Kalendar R. Comprehensive web-based platform for advanced PCR design, genotyping, synthetic biology, molecular diagnostics, and sequence analysis. Molecular Therapy Nucleic Acids, 36(4): 102716.
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2024
Kalendar R, Kairov U. Genome-wide tool for sensitive de novo identification and visualisation of interspersed and tandem repeats. Bioinformatics and biology insights, 18: 1-11.
DOI PDF -
2024
Kalendar R, Shevtsov A, Otarbay Z, Ismailova A. In silico PCR analysis: a comprehensive bioinformatics tool for enhancing nucleic acid amplification assays. Frontiers in Bioinformatics, 4: 1464197.
DOI PDF -
2022
Kalendar R, Shustov AV, Akhmetollaev IA, Kairov U. Designing allele-specific competitive-extension PCR-based assays for high-throughput genotyping and gene characterization. Frontiers Molecular Biosciences, 9:773956.
DOI PDF -
2022
Kalendar R, Baidyussen A, Serikbay D, et al. Modified 'Allele-specific qPCR' method for SNP genotyping based on FRET. Frontiers in Plant Science, 12:747886.
DOI PDF -
2022
Kalendar R. A guide to using FASTPCR software for PCR, in silico PCR, and oligonucleotide analysis. Methods in molecular biology, 2392: 223-243.
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2021
Kalendar R, Kospanova D, Schulman AH. Transposon-based tagging in silico using FastPCR software. Methods in molecular biology, 2250: 245-256.
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2017
Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI. FastPCR: an in silico tool for fast primer and probe design and advanced sequence analysis. Genomics, 109: 312-319.
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2017
Kalendar R, Tselykh T, Khassenov B, Ramanculov EM. 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.
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2014
Kalendar R, Lee D, Schulman AH. FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. Methods in Molecular Biology, 1116: 271-302.
DOI PDF -
2011
Kalendar R, Lee D, Schulman AH. Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics, 98(2): 137-144.
DOI PDF
● Key References
Thermodynamic parameters used in primer/probe calculations, and the published methods the individual tools implement
- Hoess RH, Ziese M, Sternberg N 1982. P1 site-specific recombination: nucleotide sequence of the recombining sites. PNAS, 79(11):3398-3402. — the loxP site as two 13 bp inverted repeats around an 8 bp spacer, which is the structure the loxP/FRT tool is built on.
- Hoess RH, Wierzbicki A, Abremski K 1986. The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res, 14(5):2287-2300. — spacer homology between the two sites is what efficient recombination requires, and sites with symmetric spacers excise and invert with equal frequency; the lox511 spacer comes from here.
- Schlake T, Bode J 1994. Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry, 33(43):12746-12751. — the same rule for Flp, and the source of both F3 and F5: each recombines with its own kind but is impervious to a wild-type FRT site.
- Lee G, Saito I 1998. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene, 216(1):55-65. — the systematic single- and double-substitution spacer set, and the source of both lox2272 and lox5171: each recombines with its own kind but not with the other, nor with wild-type loxP. Matched spacers alone do not guarantee wild-type efficiency, which is why the tool calls compatibility and not efficiency.
- Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW 2007. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature, 450(7166):56-62. — loxP, lox2272 and loxN used as a mutually incompatible set, and both outcomes the tool predicts put to work: excision between like sites, inversion between opposed ones.
- Albert H, Dale EC, Lee E, Ow DW 1995. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J, 7(4):649-659. — the arm mutants the tool carries as lox66 and lox71. Each changes five bases of one arm and keeps the wild-type spacer, so recombining the pair hands both mutated arms to one product site, which binds Cre poorly and makes the reaction effectively one-way.
- Shaw D, Serrano L, Lluch-Senar M 2021. Lox'd in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments. Microbiology, 167. — the survey that found 17% of papers had lox66 and lox71 the wrong way round, because the name a mutant earns depends on the direction its site is written. This tool's assignment, and its habit of printing the matched sequence beside the name, follow from this paper.
- Sauer B, McDermott J 2004. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res, 32(20):6086-6095. — Dre and its 32 bp rox site, 14 + 4 + 14 rather than 13 + 8 + 13. Dre does not act on lox nor Cre on rox, which is why the compatibility table requires the same enzyme before it looks at the spacer.
- Tesson L, Heslan JM, Ménoret S, Anegon I 2002. Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR. Transgenic Res, 11(1):43-48. — zygosity from the copy-number ratio of the insert against a single-copy reference gene. This is the question the genotyping tool deliberately does not answer: its assay pair reads presence and absence, so a multi-copy integrant reads as a homozygote. See its scope notes.
- Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA 2008. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry, 47(19): 5336-5353.
- Watkins NE, SantaLucia JJ 2005. Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res, 33(19): 6258-6267.
- Owczarzy R, You Y, Moreira BG, Manthey JA, Huang L, Behlke MA, Walder JA 2004. Effects of sodium ions on DNA duplex oligomers: Improved predictions of melting temperatures. Biochemistry, 43(12): 3537-3554.
- McTigue PM, Peterson RJ, Kahn JD 2004. Sequence-Dependent Thermodynamic Parameters for Locked Nucleic Acid (LNA)-DNA Duplex Formation. Biochemistry, 43:5388-5405.
- Allawi HT, SantaLucia J Jr 1997. Thermodynamics and NMR of internal G·T mismatches in DNA. Biochemistry, 36:10581-10594. — source of the DNA nearest-neighbour parameters used for melting-temperature calculation.
- SantaLucia J 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS, 95:1460-1465.
- Xia T, SantaLucia J Jr, Burkard ME, Kierzek R, Schroeder SJ, Jiao X, Cox C, Turner DH 1998. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry, 37(42):14719-35.
- Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr 1999. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A·A, C·C, G·G and T·T mismatches. Biochemistry, 38:3468-3477. — internal single-mismatch parameters used by the TSDR probe designer.
- SantaLucia J Jr, Hicks D 2004. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct, 33:415-440.
- Zhang DY, Winfree E 2009. Control of DNA strand displacement kinetics using toehold exchange. J Am Chem Soc, 131(47):17303-17314. — toehold-exchange kinetics model behind the TSDR designer.
- Saitou N, Nei M 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 4(4):406-425. — tree-building method used by the phylogenetics tool.
- Felsenstein J 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39(4):783-791. — bootstrap support values.
- Kimura M 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol, 16:111-120. — two-parameter distance correction.
- Jukes TH, Cantor CR 1969. Evolution of protein molecules. In: Mammalian Protein Metabolism, Academic Press, pp. 21-132. — one-parameter distance correction.
- Zhang DY, Chen SX, Yin P 2012. Optimizing the specificity of nucleic acid hybridization. Nat Chem, 4:208-214. — near-thermoneutral probe design principle used by the TSDR designer.
- Hsu PD, Scott DA, Weinstein JA, et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31:827-832. — position-weight off-target model used by the CRISPR guide designer.
- Doench JG, Fusi N, Sullender M, et al. 2016. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol, 34:184-191. — Rule Set 2 / CFD; deliberately not reimplemented here, see the guide designer's scoring notes.
- Zetsche B, Gootenberg JS, Abudayyeh OO, et al. 2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163:759-771. — Cas12a PAM and staggered-cut geometry.
- Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE 2016. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol, 34:339-344. — ssODN strand choice and the 36/91 nt asymmetric donor.
- Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533:420-424. — cytosine base editing window.
- Gaudelli NM, Komor AC, Rees HA, et al. 2017. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551:464-471. — adenine base editing window.
- Anzalone AV, Randolph PB, Davis JR, et al. 2019. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576:149-157. — prime editing; pegRNA PBS/RTT design rules and PE3/PE3b nicking.
- Nelson JW, Randolph PB, Shen SP, et al. 2022. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol, 40:402-410. — tevopreQ1 3′ motif (epegRNA).
- Gootenberg JS, Abudayyeh OO, Lee JW, et al. 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356:438-442. — SHERLOCK; synthetic-mismatch crRNA for single-base discrimination.
- Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. 2018. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 360:439-444. — SHERLOCKv2; orthologue multiplexing and lateral-flow readout.
- Chen JS, Ma E, Harrington LB, et al. 2018. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360:436-439. — DETECTR; ssDNA collateral reporter.
- Piepenburg O, Williams CH, Stemple DL, Armes NA 2006. DNA detection using recombination proteins. PLoS Biol, 4(7):e204. — recombinase polymerase amplification (RPA).
- Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL 1995. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49-53. — polymerase cycling assembly, the method behind the PCA gene assembly tool.
- Hoover DM, Lubkowski J 2002. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res, 30(10):e43. — the global optimisation of a whole oligo set that the PCA tool deliberately does not attempt; see its scope notes.
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