Design primers for PCR from given DNA sequence

Question

Given the sequence: 5’-ACTGACTATGTAGAA………GGCCCTAAGGGCCAA-3’ (1)

You wish to do PCR of this dsDNA to add “overhangs” on the ends of it. On the 5’ end of you will put the restriction site for NdeI enzyme (5’-CATATG). On the 3’ end you will put the restriction site for MspI (5’-CCGG).

Write the sequences for the “forward” and “reverse” primers to do this. Each primer should be 16-18 nt, with 12 nt for hybridization and 4-6 nt for “overhang.”

I am a little confused as to how to write the forward and reverse primers for the sequence. From videos I have seen that I should write the complementary strand of my giving strand. Which would be:

3' -TGACTGATACATCTT........CCGGGATTCCCGGTT-5' (2)

Then from sequence (1) I should get the reverse primer and from sequence (2) the forward primer. However, this doesn't allow me to add both restriction enzymes because both primers would be from 5' to 3'.

Or should I simply take the given sequence and write the primers only from that? So that the primers would be:

Forward primer: 3’-GGCCTGACTGATACAT

Reverse primer: GGATTCCCGGTTGTATAC - 5’

where the bold nts are the restriction sites.

Thank you!

Answer 1

As alluded to in the comments above, remember that you are amplifying double-stranded DNA, so two new reverse-complementary strands should be generated from each cycle (so that they can hybridize with eachother). As written in your answer, both of your primers would hybridize with your given strand, and the amplicons would not overlap (one binds at each end, but both would amplify in the same direction).

To fix this, you need one primer that binds to the target sequence, and one that binds to the reverse complement strand, which you have already generated. The new amplicons extend 5' -> 3' (from the 3' end of the primer not the template), so your primers need to bind at the 3' end of their respective strand. This link has a good visualization of that.

The 3' end of the primer needs to hybridize tightly with the template, since this is where extension happens (discussed in this guide). So your flanking restriction site sequences will always be on the 5' end of either primer (notice that the question actually hints at this by indicating the 5' end of each flank).

The primer given as your "reverse" primer will actually bind to the 3' end of your given sequence in the appropriate orientation, but you may want to put your 3' flank onto the 5' end of that primer sequence, as this will be theoretically be the 3' end of your amplicon. Your other primer will need to bind the 3' end of your reverse complement (which is the 5' end of your target), so it will have the 5' restriction site flank.

By convention, primer sequences are always given in 5'---3' format. So make sure your answer follows that convention. (i.e. 5'-Flank-primer-3').

If all of this sounds really confusing, that's because it is. I do this for a living, and I still always double check my primers in a program like Primer3