Based on the most up-to date nearest neighbor thermodynamic data, Oligo's search algorithms find optimal primers for PCR, including TaqMan, highly multiplexed, consensus or degenerate primers. Multiple file batch processing is possible. It is also an invaluable tool for site directed mutagenesis. For each primer or primer pair, Oligo's various analysis windows show a multitude of useful data, such as DNA and RNA secondary structure, dimer formation, false priming and homology, internal stability, composition and physical properties.
With Oligo you can analyze open reading frames down to predicted molecular weight and pKa of proteins, and search for restriction enzyme sites, not only in DNA but also in reverse-translated proteins. The first version of the software appeared on the market in It went through several modifications, and the last one, the change from version 6 to 7, was the most comprehensive.
Oligo search protocols scoring system may be customized in detail, so you may optimize the results according to your specific needs. Oligo runs on Macintosh and Windows and it may be downloaded from this site. Increasing this number can increase the chance of finding a specific primer pair but the process will take longer.
The maximum number of PCR targets amplicons to be shown when designing new primers. The maximum number of PCR targets amplicons to be shown when checking specificity for pre-designed primers. The maximum number of PCR targets amplicons to be found on any single sequence in the search database. The number of consecutive Gs and Cs at the 3' end of both the left and right primer.
The maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer. The option "Use Thermodynamic Oligo Alignment" instructs Primer3 to use thermodynamic alignment models instead of old traditional secondary structure alignment for calculating the propensity of oligos to form hairpins and dimers while the option "Use Thermodynamic Template Alignment" instructs Primer3 to use thermodynamic alignment models instead of old traditional secondary structure alignment for calculating the propensity of oligos to anneal to undesired sites in the template sequence.
Or mark the source sequence with: Overlap junctions [?
This requires that the left or the right primers to span a junction that is just 3' of any such positions. For example, entering "50 " would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position and counting from 5' to 3'. You can also specify in the fields below the minimal number of nucleotides that the left or the right primer must have on either side of the junctions. This option is useful if you want a primer to a span specific junction on the template.
Primer3 uses this argument to calculate oligo melting temperatures. Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al. According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations.
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The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if Concentration of divalent cations is specified. Option for specifying the salt correction formula for the melting temperature calculation. There are three different options available: Schildkraut and Lifson , DOI: The default value of Primer3 version 1.
SantaLucia , DOI: Owczarzy et al. Option for the table of Nearest-Neighbor thermodynamic parameters and for the method of melting temperature calculation.
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Two different tables of thermodynamic parameters are available: Breslauer et al. The nanomolar concentration of annealing oligos in the PCR. Note that this is not the concentration of oligos in the reaction mix but of those annealing to template. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation ii Nucleic Acids Research, vol 18, num 21 where a suitable value for a lower initial concentration of template is "empirically determined".
The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template including PCR product in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.
With this option on, the program will automatically retrieve the SNP information contained in template using GenBank accession or GI as template is required and avoid choosing primers within the SNP regions. If the default "Automatic" setting is selected, the program will automatically select the repeat database using the following rules. If a repeat database is available from the same organism as specified in the "Organism" field by user see above , then that repeat database will be used.
For example, if "Human" is specified, then the human repeat database will be selected.
If a repeat database from the same organism is not available, the database from the closest parent of that organism in the taxonomy tree will be selected. For example, the rodent repeat database will be selected if "Mouse" is specified in "Organism" field. However, no repeat database will be selected if "Gallus gallus" is specified since a repeat database from its taxonomical parents is not available. This option enables our new graphic view which offers much more details for your template and primers. It will replace the current graphic view in the future.
Reset page Save search parameters Retrieve recent results Publication Tips for finding specific primers. It is highly recommended to use refseq accession or GI rather than the raw DNA sequence whenever possible as this allows Primer-BLAST to better identify the template and thus perform better primer specificity checking. A template is not required if both forward and reverse primers are entered below.
The template length is limited to 50, bps.
Primer Design with Oligo Primer Analysis Software v. 7
If your template is longer than that, you need to use primer range to limit the length i. From To. Forward primer. Reverse primer. Min Max.
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Min Opt Max Max T m difference. No preference Primer must span an exon-exon junction Primer may not span an exon-exon junction [?
Exon at 5' side Exon at 3' side. Primer pair must be separated by at least one intron on the corresponding genomic DNA [? Enable search for primer pairs specific to the intended PCR template [? Automatic User guided No user guidance [? Primer must have at least 1 2 3 4 5 6 total mismatches to unintended targets, including at least 1 2 3 4 5 6 mismatches within the last 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 bps at the 3' end. Ignore targets that have 1 2 3 4 5 6 7 8 9 or more mismatches to the primer.
Show results in a new window Use new graphic view [? Advanced parameters Note: Parameter values that differ from the default are highlighted in yellow Primer Pair Specificity Checking Parameters Max number of Blast target sequences 10 50 [? Min Opt Max.
Primer Pair. Any 3'. For thermodynamic alignment model only. Max Template Mispriming Primer Pair. Schildkraut and Lifson SantaLucia Owczarzy et. Primer binding site may not contain known SNP [?