Hi, you can firstly read guidelines about primer design (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html). If you want to check the primer's secondary structure you can use the Oligoanalyzer tool or Netprimer. Second one is better because you can set the anneling temperature.

I do not want to make publicity but I find that Primer3 ALWAYS makes good primers http://bioinfo.ut.ee/primer3-0.4.0/primer3/

Dear Arpita,

I agree with Marek that you should read the guidelines to design the primers first to get the idea for this. Then, you should have the rol A DNA sequence because it will be used as a  template for primer design. I'm not sure whether you have your own rol A gene sequence or not but if you don't have it, you can retrieve the sequences from GenBank. If you have your own rol A gene sequence from your strain, then as Hugo mentioned above, you can use Primer3 software (this software works best for me too) or any other free software to design your primer. But, if you're going to design the primers based on the gene sequences from GenBank, it would be good if you can find the conserved region using multiple sequence alignment and use this region as your base for primer design.

Thank You all for your positive replies

The following rules may help 

A simple set of rules for primer sequence design

Primer design is an art

The following rules may guide us in designing primers.

1. Primers are 17-28 bases in length.

2. Melting Temperature (Tm):

Melting temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. Tms between 55-80oC are preferred.

The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5oC or more can lead no amplification.

Note: the annealing temperature used during PCR preferred to be 5-10 oC less than the Tm.

3. The 3' end is the end which extends:

Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming.

4. G/C content and Polypyrimidine (T, C) or polypurine (A, G) stretches:

The base composition of primers should be between 45% and 55% GC.

The primer sequence must be chosen such that there is no Poly G or Poly C stretches that can promote non-specific annealing. Poly A and Poly T stretches are also to be avoided as these will “breath” and open up stretches of the primer-template complex. This can lower the efficiency of amplification.

Ideally the primer will have a near random mix of nucleotides, a 50% GC content and be ~20 bases long. This will put the Tm in the range of 56°C – 62°C(1).

 5. Discard candidate primers that show undesirable self-hybridization:

 Primers that can self-hybridize will be unavailable for hybridization to the template. Generally avoid primers that can form 4 or more consecutive bonds with itself, or 8 or more bonds total. Example of a marginally problematic primer:

       5'-ACGATTCATCGGACAAAGC-3'

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3'-CGAAACAGGCTACTTAGCA-5'

Primers with 3' ends hybridizing even transiently will become extended due to polymerase action, thus running the primer and generating false bands. Be somewhat strict in avoiding 3' dimers. For example, the following primer self-dimerizes with perfect 3' hybridization on itself:

5'-CGATAGTGGGATCTAGATCCC-3'

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         3'-CCCTAGATCTAGGGTGATACG-5'

Primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided

.6. Product position:

Primer can be located near the 5' end, the 3' end or any where within specified length. Generally, the sequence close to the 3' end is known with greater confidence and hence preferred most frequently.  

To make sure that your primer binds not to the genomic sequence but to the coding sequence, try to make your primer include the exon boundaries.

 7. Choosing among candidate primers:

 If at this point you have several candidate primers, you might select one or a few that are more A-T rich at the 3' end. These tend to be slightly more specific in action. You may want to use more than one primer, maximizing the likelihood of success. If you have no candidates that survived the criteria above, then you may be forced to relax the stringency of the selection requirements. Also, you can use nested (internal) primers to have more accurate results. Ultimately, the test of a good primer is only in its use, and cannot be accurately predicted by these simple rules.