Parameter Details and FAQs


Input Sequence

The input sequence is any DNA sequence for which primers are to be designed. BISR Primer considers the sequence direction from 5'->3' end, so kindly make a check of sequence directionality before uploading or paste of input sequence. At present, the length of the input DNA sequence is restricted to 3000 bp. If your sequence is large then you need to use the specific range option to limit the sequence.
If you know the Genbank accession number of any sequence, then just paste the number, the programme will download the sequence online for you.

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Specific Region

Enter the position ranges if you want the primers to be located on the specific sites. The positions refer to the base numbers on the plus strand of your template (i.e., the "From" position should always be smaller than the "To" position for a given primer). Partial ranges are allowed. For example, if you want the PCR product to be located between position 100 and position 1000 on the template, you can set forward primer "From" to 100 and reverse primer "To" to 1000 (but leave the forward primer "To" and reverse primer "From" empty). Note that the position range of forward primer may not overlap with that of reverse primer.

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Number of Returns

This option provides the flexibility in the number of primers to be shown in the output. The default value is 20. Users can select any value from the option of 5, 10, 20, 50, 100 and all.

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Product Size

This parameter allows user to select the product size which is the length of the amplicon. The default is set to 50 to 1000. User can select the lower range of 50 to upper range of 900. User can select any suitable option from the given range. But remember shorter size of product will be calculated faster than the longer size.

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Product Ta

Product Ta refers to the annealing temperature at which primer binds to the template.It depends on the length and GC content of the primers. In general, the Ta should be 5 °C below the lowest Tm of the pair of primers. The low Ta will enhance the secondary dimerization which leads to non-specific amplification. On the other side, a very high value of Ta will cause the reduction in primer annealing and thus result in low amplification. The default value = 55.0 ± 5.0 °C.
The Ta was calculated using the Rychlik formula :

Ta = [0.3 x Tm(primer)] + [0.7 x Tm(product)] - 14.9

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Primer Length

This allows user to select the primer length of both left and right primers. The length of the primer is one of important factor in DNA amplification. The length should not be very short or very long. It should be such that it favors high binding specificity and low secondary structure formation and dimerization. The given range is from 18bp to 24bp.

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Primer Tm

This allows user to select the primer Tm which is one the most important parameter in designing PCR primer and during the experiment. Here user can set the min and max value of Tm value of primer. Given several methods, user can select any method. The default method is Santa Lucia(1998) including salt correction parameter suggested by Owczarzy et al.(2004).
The melting temperature(Tm) in PCR cycle is that temperature at which half of the primers (50%) present in a reaction becomes single stranded means dissociate themselves from the DNA duplex (primer-template annealing). In contrary, it is that temperature at which half of the primer has found to annealed to the template. Basically, primer Tm represents or measures the stability of duplex.
It has been reported that Tm between 55 and 65 °C works best in most of the conditions.
Primer Tm above 65-70 ° C may lead to mispriming and cause noisy sequence. Similarly, low Tm may not work at higher temperature.
Several models have been proposed for primer Tm calculation. Among all, the thermodynamics based nearest neighbor method (Santa Lucia, 1998) found to be the most accurate and reliable for Tm calculation.

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GC Percent(%)

The percentage of GC plays significant role in binding specificity of primer with template. The G-C bond is strong than A-T bond. The presence of high number of G or C in sequence favors strong primer-template binding. The default value = 30.0 - 80.0%.

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Maximum primer dimerization score

Dimerization, means the left and right primers combined to form a dimer which causes very poor or no yield of the PCR product. So it is essential to avoid this dimerization condition of primers.
The dimerization score represents maximum allowable local alignment score between left and right primers. The local alingnment score was calculated using scoring matrix where match, mismatch and gap value were set to +1, -1 and -2. The higher the score poor will be the resulting primers and may form the dimer. The default value is set to 8, which represents the upper limit of local alignment score, thus, primer-pairs having score higher than 8 will be discarded by the process.

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Maximum 3' self complementarity

The self complementarity means the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. 3' end of primer ought to have a strong binding with template which promotes the chain elongation. Hence, it is important that the 3' end of a primer should not show any binding affinity against the entire sequence of other primer. Here, the Maximum 3' self complementarity score represents the maximum allowable limit of 3' selef complementarity. Higher score tends to improve the chance of self annealing between forward and reverse primers. The default score value is set to 3.

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Maximum poly X Repeats (allowed limit)

Maximum poly X Repeats represents the maximum limit of allowable repeat of any base. The default value is set to 3. So the repeat of any three consecutive bases are allowed( Eg. AAA).

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3' GC Clamp (minimum limit)

GC Clamp represents the presence of consecutive GC at the 3'end of a primer. It improves the binding stability at 3'end which is necessary for longer amplication. Higher the number of GC at 3'end more would be the thermodynamic stability. The default value is set to 'All' which means all the primers without or with GC clamp are allowed. User may restrict the primers with specific number of GC Clamp. Imposing the GC clamp restriction may drastically reduce down the number of primers. You may use the restriction when you get the large numbers of primers. Also, in the process, we removed the presence of three consecutive G or C at 3'end.

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Salt Concentration(millimolar)

The millimolar concentration of salt (usually KCl) in the PCR. The program uses this argument to calculate oligo melting temperatures. Default is 50.0 mM.

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Annealing Oligo Concentration (50 nM)

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.Program uses this argument to calculate oligo melting temperatures.
The default (50nM) works well with the standard protocol used at the BISR for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius.
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.

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Allowed hairpin forming bases (minimum limit)

Hairpin represents the secondary structure formation in the primer. These secondary structures can also be the bulge and loops. Here we have set the minimum limit of allowed hairpin bases to 2. We discared the primers in which more than allowed bases are forming the hairpin.

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Avoid T base at 3' end (select allowed limit)

It has been reported that the presence of more T at the 3'end improves the thermodynamic instability of primers. Users may use this option to restrict the number of T bases at the 3'end.

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Avoid positional proximity between primers

This option avoids the positional proximiting between primers which means it take cares of the distance between two primer positions. It should not be too close or consecutive in nature.

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