However this will increase the search time. The maximum number of candidate primer pairs to screen in order to find specific primer pairs The candidate primers are generated by primer3 program. 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 : e. Enter a list of space separated nucleotide positions. 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. Minimal number of nucleotides that the left or the right primer must have at the 5' or 3' side of the junctions Concentration of monovalent cations Help The millimolar concentration of salt usually KCl in the PCR.
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.
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: 1. 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.
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. Retrieve recent results Publication Tips for finding specific primers Reset page Save search parameters. 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. If your template is longer than that, you need to use primer range to limit the length i. Range Help Clear. From To.
Forward primer. Reverse primer. Help Optionally enter your pre-designed forward primer. Help Optionally enter your pre-designed reverse primer. Min Max. Min Opt Max Max T m difference. No preference Primer must span an exon-exon junction Primer may not span an exon-exon junction Help This controls whether the primer should span an exon junction on your mRNA template.
Min 5' match Min 3' match Max 3' match. Enable search for primer pairs specific to the intended PCR template Help With this option on, the program will search the primers against the selected database and determine whether a primer pair can generate a PCR product on any targets in the database based on their matches to the targets and their orientations.
Automatic User guided No user guidance Help Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets.
Entrez query optional Help You can use a regular entrez query to limit the database search for primer specificity.
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. Help This requires at least one primer for a given primer pair to have the specified number of mismatches to unintended targets.
Ignore targets that have 1 2 3 4 5 6 7 8 9 or more mismatches to the primer. Help This is another parameter that can be used to adjust primer specificity stringecy. Show results in a new window Use new graphic view Help This enables our new graphic display that offers enhanced overview for your template and primers.
Note: Parameter values that differ from the default are highlighted in yellow Advanced parameters Primer Pair Specificity Checking Parameters Max number of sequences returned by Blast 10 50 Help Maximum number of database sequences with unique sequence identifier Blast finds for primer-blast to screen for primer pair specificities. Help The maximum number of PCR targets amplicons to be shown when designing new primers. Help The maximum number of PCR targets amplicons to be shown when checking specificity for pre-designed primers.
Help The maximum number of PCR targets amplicons to be found on any single sequence in the search database. Min Opt Max. Help The number of consecutive Gs and Cs at the 3' end of both the left and right primer. Help The maximum stability for the last five 3' bases of a left or right primer.
Help The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer. The process of designing specific primers typically involves two stages. First, the primers flanking regions of interest are generated either manually or using software tools; then they are searched against an appropriate nucleotide sequence database using tools such as BLAST to examine the potential targets.
However, the latter is not an easy process as one needs to examine many details between primers and targets, such as the number and the positions of matched bases, the primer orientations and distance between forward and reverse primers.
The complexity of such analysis usually makes this a time-consuming and very difficult task for users, especially when the primers have a large number of hits. Furthermore, although the BLAST program has been widely used for primer target detection, it is in fact not an ideal tool for this purpose as BLAST is a local alignment algorithm and does not necessarily return complete match information over the entire primer range.
This tool combines BLAST with a global alignment algorithm to ensure a full primer-target alignment and is sensitive enough to detect targets that have a significant number of mismatches to primers.
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