Primer design - (Apr/12/2011 )
I got a sequence from NCBI and the sequence showed completed CDs around 750aa (more than 2000bp). What does that mean? Is CDs same with cDNA sequence. Is this CD sequence exclude intron and can I use CD sequence to design a primer?
How can I design a primer using CD sequence? should I choose the reverse primer as near as to 3'end?
The CDS is the protein "coding sequence" of a gene, so for most intended purposes you can assume that the CDS is the same as the cDNA. Sometimes the CDS may contain other sequences, such as UTRs, but for the most part they should be the same.
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.
This is the sequence database that I got from NCBI : Lycopersicon esculentum ethylene receptor homolog (ETR5) mRNA, complete cds. What does it mean? THe origin sequence is 3670bp and when I clicked on CDs, the sequence is 2304bp. So, where can I get the mRNA sequence. ANd how can I design the primer? should I just use default setting on primer3 database?
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??