PerlPrimer - open-source PCR primer design

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Primer melting temperature calculations

Primer melting temperature and primer-dimer dG°37 calculations are based on the thermodynamic nearest-neighbour method, using values given by the following papers:

Allawi HT, SantaLucia J Jr. Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry. 1997 Aug 26;36(34):10581-94

SantaLucia J Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5.

Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry. 1999 Mar 23;38(12):3468-77.

Allawi HT, SantaLucia J Jr. Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects. Biochemistry. 1998 Jun 30;37(26):9435-44.

Allawi HT, SantaLucia J Jr. Thermodynamics of internal C.T mismatches in DNA. Nucleic Acids Res. 1998 Jun 1;26(11):2694-701.

Allawi HT, SantaLucia J Jr. Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA. Biochemistry. 1998 Feb 24;37(8):2170-9.

The above papers allow the calculation of Tm for 1M NaCl. To allow for relevant salt conditions, the formula developed by von Ahsen, et al. (2001) is used to adjust the calculated entropy.

von Ahsen N, Wittwer CT, Schütz E. Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas. Clinical Chemistry. 2001; 47(11):1956-1961

Bisulphite PCR conditions

Conditions for Bisulphite sequencing are based upon Warnecke, et al (2002) as well as other guidelines provided by Susan Clark.

Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clarke SJ. Identification and resolution of artifacts in bisulfite sequencing. Methods 2002; 27:101-107.

CpG Island detection

PerlPrimer uses an algorithm based on Gardiner-Garden and Frommer (1987) to detect CpG islands in sequences. By default, to be considered an island a region of DNA should have a minimum GC content >= 50% and a minimum (observed CpG dinucleotides)/(expected CpG dinucleotides) >=0.6, as calculated from the average of 10 windows of 200 base pairs, and these conditions should hold true over a minimum of 200 base pairs. Note that these parameters may be changed in the preferences.

Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol. 1987 Jul 20;196(2):261-82.

An option is also provided to emulate the program cpgplot. Please note that the same parameters will give a different result when this option is checked as cpgplot overestimates the calculated %GC and obs/exp by a factor of 11/10 (briefly, cpgplot incorrectly takes an average of 10 windows by summing 11 windows and dividing by 10). The result is that island boundaries will be somewhat larger when using this option.