Today we actually started labs 3 and 4, which will be finished tomorrow.
Lab #3
Lab 3 is very similar to Lab 2, except for the fact that this time we ACTUALLY set up the Restriction Enzyme Digestions (REDs), instead of simply running pre-cut DNA in a gel. We added DNA, restriction buffer, and restriction enzymes to a mix, and incubated it for 30 minutes at 37ºC. Why that temperature? The enzymes have been isolated from bacteria that live in mammals and, as we know, tht is our body temperature. That means that the enzymes will be most efficient at that temperature. They will still work at lower temperatures, but they would take a lot longer to do the same job. Since we incubated them at 37º, I stored them in the fridge (4º), where the enzymes won't act almost at all. If we had not incubated the digestions, they should be sitting at room temperature until tomorrow morning.
Tomorrow we will run a gel electrophoresis with such samples and compare them with the ones we ran last week in lab#2. Results should be pretty similar. Yes, you'll have to estimate the size of the DNA contained in the bands, using the visual method and the standard curve using semi-log paper. I'll e-mail you a semi-log paper pdff file in case you want to print some more copies.
Lab 4/5 - PV92 PCR
PV92 is a locus found in chromosome 16 in humans. As you should remember, we all have two copies of each chromosome, so we have two chromosomes 16, and therefore we have two copies of the PV92 locus.
What does PV92 do? Nothing! It does not code for any thing, just as the 95% of the human genome. Only about 5% of our genome actually encodes some sort of product. This locus was named in the old days of karyiotyping, when we didn't really know what different areas stained in a chromosome did. After we sequenced the human genome, we figured out that the names given to many loci didn't actually correspond to a functional gene. PV92 may be a pseudogene (a duplicated gene that lost its function), so for practical purposes we'll consider it a gene..
The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. And therefore when we think about mating in primate populations, it is random when it comes down to PV92. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population, so PV92 has been used for a lot of human population genetics studies.
There is another nice thing about PV92. Some people have an Alu sequence inserted in the very middle of the PV92 locus. This Alu insert, an example of an intron (a DNA fragment found in between coding regions of a gene, that doesn't code for any thing and that it's spliced during transcription in functional genes), makes pV92 300 bases longer, which is a difference big enough that it can be visualized in a gel (originally PV92 is about 600 base pairs long).
An Alu sequence is a retrotransposon (a DNA fragment that has been inserted in a location where it doesn't belong via reverse transcription, i.e. mRNA actually writes a sequence into the genome) found throughout primate genomes. There are thousands of copies of Alu sequences found in a single genome.
PCR, the Polymerase Chain Reaction, a method to amplify (make many copies of) specific DNA fragments, is used to generate enough DNA that it can be studied with a variety of thechniques. Using PCR we will amplify our PV92 loci to see if we have the Alu sequence insert in any of our copies of PV92. Each one of us may have 0, 1, or 2 alu inserts in our PV92 copies and we will find out through PCR and gel electrophoresis.
Today we extracted our own DNA. We scraped our cheeks to get some epithelial cells, we neutralized ions (with the InstaGel) that may be used as cofactors by DNases to chop out DNA, and then we bursted our cells open (with the water baths) to extract the DNA from the nuclei.
After lab I added your DNA and mine, plus three positive controls, to a master mix with all the constituents of a PCR: buffer, nucleotides, Taq polymerase, magnesium, and primers (synthesized single stranded oligonucleotide sequences) and put the tubes into the thermocycler. We'll talk about details of how PCR works later.
Tomorrow we'll run a gel electrophoresis to figure out who among us has the Alu insert in his/her PV92 locus, and if it is a single copy or two.
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