Question

Can you please explain how to get the primers?

Answer 1

Ans

A short sequence of DNA or RNA that work as a starting point for DNA synthesis is called Primer. In the cell, the RNA primer utilized to initiate DNA replication on the both leading and lagging strands, because of the DNA polymerase enzyme can only add new nucleotides to an existing strand of DNA i.e DNA primer is not present in vivo in humans.

In vitro (outside the cell) DNA synthesis using DNA primers because they are more stable for temperature.

Presently, it is good to pick up published primers from reputed journals, if we don't find any, for that

1. Check out the sequence of desired gene from NCBI database

2. Submit that sequence in the primer3 tool, go for pick primer option.

3. It provides a set of primers

4.Check those set of primers for primer quality like primer dimer formation, hairpin loop formation(there are online tools to find out), annealing tempereature( difference between forward and reverse primer should not exceed 5^oC andd annealing temperature should be between 48-63)

5 Primer sequence should not be less than 17bp and not exceed 25bp(less than 17bp nonspecificity, more than 25bp high annealing temp)

6.GC content should not be less than 45 and more than 60.

once you pick the good primer cross check the primer using UCSC browser, or ncbi blast tool.

Website for primer design:

http://primer3.sourceforge.net/)

Primer3Plus (see website: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/)

http://www.ncbi.nlm.nih.gov/tools/primer-blast/