Lecture - Overview on chromosomes and PCR

We revisited some of the topics we covered on Monday and elaborated on some othres. We talked about how the DNA is packed in chromosomes, and how chromosomes compare accross related species.

We also covered some details of PCR, which we have been actually performing in lab... which reminds me, there is a video you should see, more than once. I recommend learning the lyrics. Yes they are cheesie, but they are useful!

They are nerds, and they obviously grew up in the 80's... oh, and it's a Bio-Rad ad, in case you haven't have enough of them with our lab kits... The PCR song.

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Lab 04 - Detecting genetic modifications in food

Bio-Rad's GMO Investigator Kit

In this lab we are trying to find evidence of genes that have been inserted into a plant's genome. There are two ways of finding out:

Testing for the presence of proteins that the trans-genes produce
Amplifying a fragment of DNA that contains the trans-gene via PCR
Since we need a lot of fresh tissue and a kit to test for several of the proteins that the most common trans-genes code for, materials that we don't have, we are using the almighty PCR.

Most trans-genes have the 35S promoter from the cauliflower mosaic virus (CMV 35S), and the nopaline synthase (NOS) terminator from Agrobacterium tumefaciens. These sequences come from totally different organisms from that used to isolate the actual trans-gene, but they are recognized by the plant cell machinery (mainly its polymerase), so they can actually produce the product that the gene is intended for. Even if the gene is in the genome, it will NOT work if there are no promoters AND terminators that the plant can recognize. Since there is a number of genes that have been inserted in crops in the U.S., it's easier to go after the promoter and the terminator when using PCR. CMV 35S and NOS have been use in over 85% of the GMOs, so if we have primers to amplify them, we don't have to worry about which gene was inserted (as long as the question doesn't involve knowing the specific gene).

We extracted DNA from vending machine snacks that we presume have been made from genetically modified crops, and from a certified non-GMO food control provided by Bio Rad, the manufacturers of the kit we use. The master mixes we used include primers for both CMV35S and NOS in the same mix, and in addition to those we are using a separate master mix with primers that will amplify a 455 bp region of the photosystem II (PSII) chloroplast gene, common to most plants. This will be a control to ensure we have successfully extracted DNA, so even non-GM plants should have the band present.

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Lab 03 - Human DNA extraction and PCR targeting the PV92 locus

Today we extracted DNA from our cheek cells, and then set up a PCR (Polymerase Chain Reaction) targeting the PV92 locus.

PV92 is found in chromosome 16 in humans. 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. For practical purposes we'll consider PV92 a gene, with a couple of "alleles" (see below).

The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. So when we think about mating in primate populations, PV92 reflects the mating patterns, since the allele frequency is not going to be incluenced by selection. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population. 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 (which could be an intron should PV92 be a real gene. An intron is 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) makes PV92 300 bases longer, which is a difference big enough to be visualized in an agarose 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.

We added our DNA, 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.

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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Lecture - Talking about chromosomes

Yesterday we started talking about chromosomes, their structure and some characteristics found after some whole genome research has been done in several organisms, including humans. On Friday we will continue with this topic and very likely will start with genome evolution.

Readings to come: p. 233-244 (chromosomes) and p. 245-260 (genome evolution)

Wednesday's lab: Extraction of human DNA and set up of the PV92 PCRs. It corresponds to "Exercise 4&5" in the old lab manual.

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