Tuesday, January 15, 2008

PCR: A Primer (Ha, Ha; Biologists Can Skip This)

PCR. Right. Amazing! Miraculous! Biological!

What is PCR, you ask? Polymerase chain reaction. In other words, replicating DNA over many times. You can start with, in theory, as little as one copy and make, in theory, as many copies as you want.

Brief DNA review: adenine, thymine, guanine, and cytosine are the bases; they always pair A/T and G/C. The beginning is called the 5' end, and the sequence is therefore directional, like an arrow. A sequence of 5'-GCATC-3' will have a complement of 5'-GATGC-3', which will meet up with it in reverse. (See

Let's say you have some DNA from a plant. This plant has a mutation in a gene, which causes its flowers to be white instead of red. Through a great deal of painful back-crossing, you know which gene this is; now you want to know what the mutation is. How?

First you want more copies of the gene to work with than 'a few', so you need to amplify it.

But DNA polymerase- the protein which takes nucleotides and sticks them together- needs two more things to work: a template, and a primer. The template is whatever piece of DNA you've put in. The primer is a short sequence complementary to some of the sequence you have; it's necessary because polymerases need a 'starter' to get them going. Let's assume the sequence of the gene is known. So you design primers on either side of the gene you're looking at, one going down the 'top' strand and the other headed the opposite way on the 'bottom' strand. That is, each primer matches up with the 5' end of the gene so that both strands have a starter bit.

The polymerase we use nowadays is thermostable: it works at high temperatures, and likewise isn't killed by heat. So to amplify a gene, you mix polymerase, template, primers, nucleotides, a buffer and a little salt to keep the enzyme happy (among other things), and... a little water. In a tube.* Then you heat it up to unstick the two DNA strands of your template, cool it down so the primers can bind, heat it up so the polymerase starts polymerizing, and repeat. And repeat and repeat. Luckily there are little machines for this.**

Now you have a zillion- actually, (2)^ number of cycles- copies of your gene. You can sequence it yourself, or send it out. Commercial dye-terminator sequencing typically costs $5/10 per 600 base pairs. In-house, radioactive will cost about 25 cents per 100 bp, though you can do dye-terminator, but the machines in dye-terminator are quite expensive. And then you know what the mutation is.

Sequencing genomes or SNPs or all manner of other things is generally done by different methods due to scale, but the principle is the same.

Next: Why PCR Revolutionized Biology

*Molecular biology: a) Clear liquids in a tube; b) Something that comes in a kit from Qiagen.

**Back in the day water baths and a great deal of patience were required, not least because the enzyme died after every round and had to be added back. Every time. Erf.