Manual Primer Design for a gene on the reverse strand

Question

My question might sound very naive and stupid but I am hopeless now. I read so many websites and pages but could not figure out this PCR primer design thing completely.

Some genes are on the reverse strand of the DNA as we can see on the certain databases. Like this:

Source: http://www.wormbase.org/species/c_elegans/gene/WBGene00019616#0de1-9g4356caf-3

So when I click and get the sequence data of that gene, it also says it's on (-) strand. My most potentially silly question is: Do I just copy paste that sequence and try to find my primers manually or do I need to get a reverse complement of that sequence to make it (+) strand so I can design my primers? This minus, plus stand and reverse complement thing confuses me a lot! And I also do not get why do we get the reverse complement of the reverse primer sequence once we design it.

Thanks for the help in advance!

Answer 1

Not a stupid question at all. I was just as confused by this as a beginner in molecular biology. You get the hang of it after a while. :)

I find it much more difficult to design primers from just one strand, especially the minus strand, so I always try to get both to work with. If you don't feel like doing that, just one strand is sufficient (plus or minus), but I would recommend at least getting the reverse complement in your case because plus strands are much easier to work with.

For a quick example, let's say I have a ten bp sequence I want to design primers for and this is my plus sequence:

5' ATAACTTCGT 3'

Now let's say I want a three bp primer. So the forward primer would simply be 5' ATA 3', that's easy. The reverse primer, if I just take it from there without flipping it, would be 5' CGT 3'. But if I put that into my PCR reaction, it will not do anything because DNA binds to it's complement strand, meaning that primer would only bind to 3' ACG 5', or 5' GCA 3'. So instead, I'm going to want 3' ACG 5' because it will bind to its compliment, the 5' CGT 3' part of my sequence. (Technical issue here, suppliers usually only give the option to write in 5' to 3', which makes sense, but you must remember to flip your reverse primer before giving it to them because it will be in 3' to 5' originally. In this case, I would ask for 5' GCA 3'.)

This is easier to visualize if I have both the plus and minus strand though, which would look like this:

5' ATAACTTCGT 3'
3' TATTGAAGCA 5'

If you have both strands like the example above, you can find both the forward and reverse primer without much difficulty.

I could give a more comprehensive answer of how this all works out during DNA replication that would involve illustrations, allegories, and maybe a few mildly humorous jokes, but I would hate to bore you unnecessarily, so let me know if you understand now or if you need more. ;)

Edit: Exactly (to your third comment I mean)! Remember, DNA is always read and replicated 5' to 3', so the forward primer will bind to the minus strand and be replicated 5' to 3', so will extend the sequence to the right. The reverse primer is the opposite, 3' to 5', so it will extend the plus strand to the left. After both of those happen, you will end up with two full strands. As promised, here is an illustration:

Now, to your question about Primer-BLAST. You do not have to enter a plus strand, but the tool is assuming that you are entering the plus strand, so you will get the right primers, it will just call them the wrong names (i.e. with the example above, it would give you 5' GCA 3' as your forward primer and 5' ATA 3' as your reverse). The forward and reverse primers are treated exactly the same, so this wouldn't throw off your PCR, but it would be incorrect nonetheless. To avoid confusion, I suggest utilizing a tool to reverse-compliment your minus sequence into plus before running it. It's rather easy to get the reverse compliment of any sequence; here is a good tool for that.

I hope this helps. :)