PCR: Are primers designed to be complementary to a DNA sequence within the template DNA or to the 3' ends of the template DNA?

Question

I was told that in order to produce primers, the DNA sequences at the 3' end of the template strand must be known, and that these primers are complementary to 3' ends of the template DNA. However, that causes me confusion when I view the following image from my college class, which show that the primers anneal to the template DNA further down the strand:

So do the primers need to be complementary to the last 20-35 bases at the 3' ends of the template DNA or simply to any portion of 20-35 bases near the 3' end?

Answer 1

You seem to be confused about the template DNA having a relevant "end". Most DNA molecules one would start with would be very long, often an entire chromosome. The 3' ends of the two strands are essentially never near the piece one is trying to amplify with PCR. So, no, the primers do not match these ends.

For PCR, you are trying to amplify a "region of interest" within the millions of bases you start with. The region is necessarily bounded by two known sequences (otherwise you have nothing to prime the DNA replication). The two known sequences define the region of interest and are the sequences one creates the two (different) primers -- the green and orange primers shown in the question's diagram. Since those two primers define the region of interest, and DNA replication will proceed in one direction (from 3' to 5'), they are exactly the two ends of the region to be replicated. These primers of course anneal very, very far from the ends of the chromosomes.