Whole books could be written on this, and probably have. I assume you can’t use primer design software, but need to design something by “eye.” I won’t hand you an answer but some general advice for “eyeball” primer design. I usually make my primers 21 to 24 bases in length, (longer if amplifying genomic DNA, shorter if plasmid DNA) and I aim to make them about half g/c and about half a/t. If it can’t be exactly half, g/c content up to 65-70% is good. It is better to favor g/c a little since they make stronger bonds to the template. I want all 4 bases to be represented if possible, and I want to avoid long runs of the same base or repeating groups of bases (like TATATATAT). The primer sequence should be “complex” so that it will be more likely to be unique to the one target you want. (I used to have software that showed the 4 bases in different colors, and I would “eyeball” the most colorful spots where there was a good mix of bases. And avoid the areas of one color.) And I often put a short run of 3 g’s or c’s at the 3’ end of the primer, because that is where the polymerase will start, and a strong association to the template at that end is really important. If possible, I will avoid runs of 3 bases in any other part of the primer (not always possible).
With these goals in mind I would look first tackle the “forward” primer upstream of the ATG. The forward primer is “easy” because you pick your spot and just copy the 21-24 bases verbatim from the sequence as written. (By convention you are seeing the “top strand” written in the 5’ to 3’ direction.) Count the different bases and see if they fit the “rules” above. Look at several possibilities and pick the one that you think best fits the rules.
The reverse primer is a little trickier, since you must take the sequence of your primer from the reverse compliment (or “bottom strand”) of the sequence as shown. This is easier if you have software that shows the sequence as double-stranded. In which case you would look at the bottom strand and read it right-to-left. If you don’t have that, you will have to write out the reverse compliment of the sequence downstream of the stop codon. Remember it is read in the opposite direction, so (for example) the reverse compliment of AAATGCG is CGCATTT. Now you have the reverse compliment written out as if it was the “top strand,” in its 5’ to 3’ direction, and you can apply the same rules as you did for the forward primer to make the reverse primer.
Now, most textbooks will say you have to check each primer for the tendency to fold over and stick to themselves (make “hairpins”) and also the tendency to stick to themselves or each other. This is most easily accomplished with software. You can try to look for such problems by eyeball but it is easy to miss something. In practice, though, it is a pretty rare coincidence for primers to make really bad (strong) hairpins or primer dimers- they will prefer to stick to the proper template if you use the proper annealing temperature.
Speaking of annealing temperature, you want to have about the same annealing temperature for both primers. You probably have an equation in a lab manual or textbook for calculating this. If you have followed the rules and made primers with about the same % GC content, they should be close enough. Again, primer design software takes this into account for you.
I have made hundreds of primers “by eyeball” in my day and most of them have worked well even if they violated one of the rules. You can never really know whether you have a good design until you try them. Even “bad” primer designs work well, sometimes.
Adding restriction ( R ) sites: After you design a primer and have it written in its 5’ to 3’ (left to right direction), add the sequence of the R site to the 5’ (left) end. Then even further to the left, add six random bases that aren’t a match to the template. They are only there to make sure the R enzyme can attach to the R site and cut near the end of the amplified sequence. It is OK to have a “tail” of bases that don’t match the template at the 5’ end of a primer, because (as mentioned above) it is the 3’end matching that is most critical to the function of the polymerase. Why add extra bases? Some enzymes don’t bind well if the R site is too close to the end of an amplified double-stranded fragment. Enzyme supplier catalogs will usually have an appendix that lists the ones you have to watch out for. However, primer synthesis is so cheap these days it never hurts to add the 6 extra bases to the 5’ end anyway.
Hope this helps- good luck!
Edited by OldCloner, 13 February 2019 - 08:46 AM.