Module 1, Lab 04 - Size exclusion chromatography (SEC)

Friday, September 17, 2010

Column chromatography is a common technique used in molecular biology to purify large macromolecules, such as proteins, by separating the components of complex mixtures. A solvent (usually a buffer) and the molecules to be separated are passed through a resin of glass beads (column bed) whose specific characteristics vary depending on the type of chromatography.

Size exclusion chromatography (SEC) is a technique in which the molecules are separated by size. The glass beads in the resin have tiny pores. When the mix is applied to the column large molecules pass quickly around the beads, whereas smaller molecules enter the pores in the beads and pass through the column more slowly. The buffer and the molecules are collected in separate tubes (fractions), so that the earlier tubes get larger molecules and the later tubes get smaller molecules.

In this exercise you will separate a mix of Hemoglobin (large molecule - 65,000 Daltons) and Vitamin B12 (small molecule - 1,350 Daltons) using a SEC column.

----------------------

Module 1, Lab 03 - PCR of the human PV92 locus

PV92 locus genotypes of Fall 2010 students

(click on pic to see a full-sized image)

----------------------------------------------------------------------------------

Thursday, September 16, 2010

The goal in this lab to introduce students to the Polymerase Chain Reaction (PCR), the most popular in vitro technique to make copies of target DNA fragments. We extracted DNA from our cheek cells and used it to set up basic PCRs.

Our target is the PV92 locus, located on chromosome 16. This locus may, or may not, have an insertion of an Alu element. Alu elements are a family of short interspersed repetitive elements (SINEs) that have mobilized throughout primate genomes for the last 65 My, by retrotransposition.

In this exercise you will find out if you have the PC92 Alu insertion in one, both, or none of your chromosomes.

There are more than 500,000 Alu elements per haploid genome in humans (about 5% of our genome). Depending on the insertion point they may be associated with some genetic diseases (e.g.some cases of hemophilia, familial hypercholesterolemia, severe combined immune deficiency, or neurofibromatosis type 1). But in most cases it has no effect on the individual's health.

Some Alu insertions are very recent and polymorphic. The most recent are human specific (HS) and such is the case of the PV92 insertion. Because the PV92 insertion is HS, polymorphic, neutral (invisible for natural selection), and easy to detect, it has been widely used in human genetic population studies, and it has been one of the markers used to support the out-of-Africa hypothesis.

So, do you have 0, 1, or 2 PV92 Alu insertions in your genome?

The following picture illustrates the possible outcomes of your PCRs:

The sample on lane 1 belongs to an individual with no PV92 Alu insertion, lane 2 to an individual with insertions in both chromosomes, and lane 3 to an individual with an insertion in one chromosome.

What is your genotype like?

In the mean time enjoy The PCR Song! Students in previous quarters have found this song useful to remember the sequence of steps in PCR... (Warning: Cheesy!)

----------------------

Lecture, chapter 4 - Genes, Genomes, and DNALecture, chapter 5 - DNA replication

Today we finished chapter 4.

We focused on the importance of supercoiling DNA so it fits in a cell (prokaryotes) or in a nucleus of a cell (eukaryotes). We discussed the mechanisms through which prokaryotic DNA is supercoiled and how eukaryotic DNA is packed in chromosomes as chromatin (in this case the term 'supercoiling' is not really applicable, but it is a useful analogy).

Then we started chapter 5, on DNA replication and we did a quick introduction to the replication fork and the elements involved: DNA and the replisome (all the enzymes involved in the DNA replication process)

----------------------

Lecture, chapter 4 - Genes, Genomes, and DNA

Today we covered most of chapter 4.

The main topic we covered was non-coding DNA. We talked about interspersed elements (LINEs, which are moderately repetitive, and SINEs, which are highly repetitive), and tandem repeats (satellites, minisatellites [or VNTRs], and microsatellites [or STRs]). We also talked about junk and selfish DNA, palindromes, hairpins, and stems and loops.

----------------------