Dear All,
I know that my question is simple but I have a problem with understanding the transcript database. In big simplification I don’t understand the strand orientation. I read that one strand is + while the other is - and it means that forward (+) strand is coding, and reverse (-) is a template. So if my gene of interest is on reverse strand, is that mean that in this case - strand is coding ? When I search a database (like for example ensembl) I have a transcript written in 5->3 direction. So can I use it to find the right primers ? Or should I have reverse complement ?
Thank you.
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written
by
madziapio •
1 answer
The initial designation in genomic DNA to + and - strands is arbitrary. Sometimes they are also called top and bottom strands, forward and reverse, and if you are old enough Watson and Crick strands. While the initial designation is arbitrary, from that point on all genes (or more generally, transcripts) are on + or - strands relative to that original designation. That means that the coding strand for some genes will be on the + strand, and for the others on the - strand. Neither strand of DNA is fully coding or fully a template strand.
If this strikes you as too arbitrary, keep in mind that it is difficulty to have a rule about + or - strands when both strands are used as coding strands for some genes. For prokaryotes an attempt was made some time ago to standardize the genomes so they always begin with dnaA gene on a + strand, but not sure whether that rule is still in place. Either way, genes (transcripts) should always be provided in 5' -> 3' direction regardless of the strand on which their coding sequence is, so it should be safe to design primers with that in mind.
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This is an oversimplification IMO. There is no global coding/template strand.
If your gene of interest is on the reverse strand, that is the coding strand for that gene. You'd design primers based on the transcript sequence, so once you get to a transcript, don't worry about which strand it is in. Just base your primer on the sequence.