Question

What are PCR primers? Briefly discuss how primers can be designed

Answer 1

What are PCR primers?
A short stretch of single strandard oligo nucleotide which initiate the DNA replication by hybriding to its correspondense sequence in invitro are called as primers. In general the PCR primers are deoxy oligonucleotide primers.

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Briefly discuss how primers can be designed.
The main important characters in the primer designing are
1. Length of the primer: The optimal length of the primer is 15-20 bp. Some reports suggested that the lenght of the primer is alos effects the amplicon length also.

2. Melting temperature:
The optimum melting temperature is 52-58 Degrees Celcius. Based on the Tm of the primer we can find the aneeling temperatrue also. If the metling temperatre is more than 65 degress, there is a chance of secondary annealing. So, to avoid this max primer are bulid with ~55 degrees of Tm.

3. GC content: The optimum GC content in the primer sequence is around 45-60%.

4. GC clamp: G and C presence in the last five bases at 3' end helps in specific binding etc promoter sepcific binding. So, based on the amplicon character, its need to be added.

5. Dimerization of the primer: Self and cross dimerzation of the primer effects the stability of the primer and pcr reaction. It avoid this, in primer desing self and cross dimerization have to be avoided.

6. Nucleotide repeats: Di or tri nucleotide repreats causes mispriming hence it should be avoided.

7. Nucleotide Runs: Single bases runs should be avoided, this may also leads to mispriming.

Now using primer desinging software with all these specific parameters you can generate the requeired primer for PCR instantly.