PrimeTaxa
Pick 16S rDNA primers/probes for a taxonomic branch using the Primer3 method.
OBJECTIVE: To find the best primers for exclusive PCR amplification of a given taxon. 		Primer3 copyright
Primer3 source code
Primer3 cautions Primer3 FAQ
Input the Operational Taxonomic Unit (OTU) identification number: (Must be included if near neighbors are to be checked for mishybridization. Even if a sequence is pasted in below.)
Example: 3673
OR
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.

MisHybe Check Depth Enter the number of near neighbors to check for mishybridization.
Include D125_wk29 related sequences for mishybe check
Pick left primer or use left primer below. Pick hybridization probe (internal oligo) or use oligo below. Pick right primer or use right primer below (5'->3' on opposite strand).

Session 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.
USE CAUTION because use of a consensus sequence may alter the position of the choosen target.
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.
USE CAUTION because use of a consensus sequence may alter the position of the choosen target.
Product Size Ranges
Pairs to Return: Max 3' Stability:
Max Mispriming: Pair Max 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: 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.

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

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

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 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

The primer3 backbone: Copyright (c) 1996,1997,1998,1999,2000,2001,2004 Whitehead Institute for Biomedical Research. All rights reserved.

Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:

  1. Redistributions must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution. Redistributions of source code must also reproduce this information in the source code itself.
  2. If the program is modified, redistributions must include a notice (in the same places as above) indicating that the redistributed program is not identical to the version distributed by Whitehead Institute.
  3. All advertising materials mentioning features or use of this software must display the following acknowledgment:
    This product includes software developed by the Whitehead Institute for Biomedical Research.
  4. The name of the Whitehead Institute may not be used to endorse or promote products derived from this software without specific prior written permission.
The reference for web-enabled primer3 is:
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
Source code for primer3 is available at: http://fokker.wi.mit.edu/primer3/

Acknowledgments

The development of Primer3 and the Primer3 web interface 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).

Web software based on scripts created by Steve Rozen
and Whitehead Institute for Biomedical Research. 

(primer3_www_OTU_specific.cgi v 0.1)