Posted By Caulimovirus on September 30, 2010
Note: this entire post, though completely factual, is a setup for a terribly geeky joke. You have been warned.
.: I’m sequencing the maize genome this week. Not the big one in the nucleus, mind you, but the smaller one in the chloroplast. The big genome is about 2.3 billion nucleotides long and would take a dedicated lab of experienced researchers to deduce, whereas the small one is only about 0.00014 billion and can be easily determined by a clumsy grad student.
“Cody,” you ask, “why are you wasting your time sequencing the maize chloroplast genome when Maier, Neckermann, Igloi, and Kössel did exactly that 15 years ago?” Because my maize is different, that’s why. At least, I hope so — that’s what the sequencing will find out for me. My maize is almost guaranteed to have some differences between the published sequence, and it’s these differences I will be taking advantage of.
.: I’ll be sequencing the little genome the old fashion way, which involves the construction of several primers. Amplifying DNA requires primer pairs: each segment needs one forward and one reverse primer (some labs use up and down, but not ours). Amplifying 140,000 nucleotides worth of DNA in ~5,000 nucleotide fragments with overlapping ends requires about 30 pairs of primers.
.: I spent Monday designing my primers. They have to be fairly similar to one another in certain characteristics (melting temperature, GC content, no secondary structures, etc.) while all being unique in sequence. To keep track of them all, simple names are given. The first pair of primers are simply “1 Forward” and “1 Reverse”, and so on down the line. The only reason I mention all this is because designing primers gives me the opportunity to occasionally write down, with perfect legitimacy, “10 Forward”.