Posted 12 April 2011 - 02:48 AM
How can I design a primer using CD sequence? should I choose the reverse primer as near as to 3'end?
Posted 12 April 2011 - 07:34 AM
You can use the CDS to design primers, but it is to your advantage to also know the mRNA sequence if you are going to design primers for RT-PCR. This is because you may want to design the primers spanning an intron-exon junction. One way to do this is by comparing the mRNA sequence to the CDS.
You can design the primers anywhere on the CDS, although ideally you do not want to locate your primers too close to any end. Designing the primers near the 3' end is a common strategy that takes into account the fact that mRNAs start to get degraded at the 3' end. In other words, to take into account any mRNA degradation in your samples, it is a good idea to amplify close to the end of the mRNA.
Posted 13 April 2011 - 12:38 AM
I have compared this sequence with its homolog sequence. Let say that I found the similarity at nucleotide 1450-1460. Should I design the primer that include these nucleotides??