Online Bioinformatic Tools - Primer3


Paste source sequence below (5'->3', string of ACGTNacgtn -- other letters treated as N -- numbers and blanks ignored). FASTA format ok. Please N-out undesirable sequence (vector, ALUs, LINEs, etc.) or use a Mispriming Library (repeat library):

Pick left primer, or use left primer:
Pick hybridization probe (internal oligo), or use oligo below:
Pick right primer, or use right primer below (5' to 3' on opposite strand):

Sequence Id: A string to identify your output.
Targets: E.g. 50,2 requires primers to surround the 2 bases at positions 50 and 51. Or mark the source sequence with [ and ]: e.g. ...ATCT[CCCC]TCAT.. means that primers must flank the central CCCC.
Excluded Regions: E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT.. forbids primers in the central CCCC.
Product Size Ranges
Number To Return Max 3' Stability
Max Repeat Mispriming Pair Max Repeat Mispriming
Max Template Mispriming Pair Max Template Mispriming

General Primer Picking Conditions

Primer Size Min: Opt: Max:
Primer Tm Min: Opt: Max: Max Tm Difference:
Product Tm Min: Opt: Max:
Primer GC% Min: Opt: Max:
Max Self Complementarity: Max 3' Self Complementarity:
Max #N's: Max Poly-X:
Inside Target Penalty: Outside Target Penalty: Note: you can set Inside Target Penalty to allow primers inside a target.
First Base Index: CG Clamp:
Salt Concentration: Annealing Oligo Concentration: (Not the concentration of oligos in the reaction mix but of those annealing to template.)
Liberal Base Show Debuging Info Do not treat ambiguity codes in libraries as consensus

Other Per-Sequence Inputs

Included Region: E.g. 20,400: only pick primers in the 400 base region starting at position 20. Or use { and } in the source sequence to mark the beginning and end of the included region: e.g. in ATC{TTC...TCT}AT the included region is TTC...TCT.
Start Codon Position:

Sequence Quality

Min Sequence Quality: Min End Sequence Quality: Sequence Quality Range Min: Sequence Quality Range Max:

Objective Function Penalty Weights for Primers

Tm Lt: Gt:
Size Lt: Gt:
GC% Lt: Gt:
Self Complementarity
3' Self Complementarity
#N's
Mispriming
Sequence Quality
End Sequence Quality
Position Penalty
End Stability
Template Mispriming

Objective Function Penalty Weights for Primer Pairs

Product Size Lt: Gt:
Product Tm Lt: Gt:
Tm Difference
Any Complementarity
3' Complementarity
Pair Mispriming
Primer Penalty Weight
Hyb Oligo Penalty Weight
Primer Pair Template Mispriming Weight

Hyb Oligo (Internal Oligo) Per-Sequence Inputs

Hyb Oligo Excluded Region:

Hyb Oligo (Internal Oligo) General Conditions

Hyb Oligo Size: Min Opt Max
Hyb Oligo Tm: Min Opt Max
Hyb Oligo GC% Min: Opt: Max:
Hyb Oligo Self Complementarity: Hyb Oligo Max 3' Self Complementarity:
Max #Ns: Hyb Oligo Max Poly-X:
Hyb Oligo Mishyb Library: Hyb Oligo Max Mishyb:
Hyb Oligo Min Sequence Quality:
Hyb Oligo Salt Concentration: Hyb Oligo DNA Concentration:

Objective Function Penalty Weights for Hyb Oligos (Internal Oligos)

Hyb Oligo Tm Lt: Gt:
Hyb Oligo Size Lt: Gt:
Hyb Oligo GC% Lt: Gt:
Hyb Oligo Self Complementarity
Hyb Oligo #N's
Hyb Oligo Mishybing
Hyb Oligo Sequence Quality

Copyright Notice and Disclaimer

Copyright (c) 1996,1997,1998,1999,2000,2001,2004,2006
Whitehead Institute for Biomedical Research, Steve Rozen, and Helen Skaletsky
All rights reserved.

Citing Primer3

We request that use of this software be cited in publications as

Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386

Acknowledgments

The development of Primer3 and the Primer3 web site was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute. under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander).

We thank Centerline Software, Inc., for use of their TestCenter memory-error, -leak, and test-coverage checker.

Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). Lincoln Stein championed the idea of making Primer3 a software component suitable for high-throughput primer design.