Lecture, Chapter 5 - DNA replication

Today we finished the DNA replication chapter.

We discussed the main differences between prokaryotic and eukaryotic chromosomes, and how the ends of eukaryotic chromosomes (telomeres) are repaired by the action of telomerases (three scientists were awarded the Nobel Prize in physiology or medicine, in 2009, because of their research on telomerases).

Among the differences between prokaryotic and eukaryotic DNA replication we mentioned how in eukaryotic chromosomes have multiple replication origins, and in their DNA replication process there are two DNA polymerases and a few proteins that are not found in the prokaryotic DNA replication.

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Lecture, chapter 5 - DNA replication

Today we covered most of chapter 5, on DNA replication. We talked about the replication fork and the replisome (all the enzymes that are part of the replication fork), including the difference in between how the leading and lagging strands are synthesized.

We also mentioned how important DNA looping is for the lagging strand to be synthesized. This video illustrates such phenomenon beautifully:

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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.

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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)

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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!)

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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)

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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.

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Module 1, Lab 01 - Phenol-chloroform DNA extraction

Today we had our first lab (what an experience!) in which students started extracting DNA from their own blood. We had the assistance of Dr. Lisa Walden and her students from ONU's Clinical Laboratory Science, who very kindly offered to do the phlebotomies necessary to obtain the samples.

The exercise took longer than expected, so we didn't finish the process, but the samples were frozen after the cell lysis step and will be ready to continue with the addition of phenol-cholorform-isoamyl alcohol (PCI) in the next lab session.

Tomorrow: Restriction Enzyme Analysis (RED) of lambda-DNA.

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Friday, September 10, 2010

Module 1, Lab 02 - Restriction Enzyme Digestion (RED) of Lambda DNA

A micrograph of multiple bacteriopages

Restriction enzymes are one of the most basic and important tools in molecular biology. They evolved in bacteria to attack and cut (cleave) foreign DNA, mostly from bacteriophages (viruses that "eat" bacteria). But hey have been isolated to be used in the lab, and are useful to cut any kind of DNA, not just viral.

Cleaving DNA is the first step in any technique that involves recombinant DNA technology. There are techniques that use special enzymes to paste (ligate) different fragments of DNA. For instance a gene can be ligated into a plasmid that can be inserted into bacteria to make many copies of it via bacterial reproduction (cloning), something we will do in a few weeks.

Today we used lambda DNA (DNA from the common lambda bacteriophage) as the substrate to be cleaved with three different restriction enzymes: EcoRI, HindIII, and PstI.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today we finished chapter 3 on the basic structure and function of DNA, RNA, and proteins.

We talked about characteristics of base pairing in DNA, characteristics and functions of different kinds of RNA, the central dogma of molecular biology, and about the very basic structure of proteins.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today we had our first lecture. We introduced the course and had an overview of the syllabus. Then we had an overview of the "road map" of this course, a series of major topics that are the backbone of what the course is going to be, and how each one of the chapters fit within such plan.

We staring covering chapter 3, on DNA, RNA, and proteins. We talked about the very basics structure of nucleotides and their components, how they are linked, and the rationale behind their nomenclature. We finished the lecture talking about the double helix structure of DNA.

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Welcome to the Introduction to Molecular Biology class, Fall 2010-11

Welcome to the intro Molecular Biology class (Biol 217) of the Fall quarter 2010-11!

In this blog you will find updates of our progress in the class, both in lecture and in the lab. You can use it as a topic guide for studying for exams and make comments when ever you see it fit.

The blog will also be used as a means of posting information relevant for the class, such as external links, occasional changes in scheduling, and general announcements.

If you have any ideas to improve the blog or about information that you would like to see posted, please send me an e-mail. Cheers and good luck this quarter!

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IV ONU Intro Molecular Biology Symposium

Department of Biological and

Allied Health Sciences

Matile Center 138

February 17 - 18, 2010

The ONU Intro Molecular Biology Symposium takes place every Fall and Winter quarters, when the Introduction to Molecular Biology (Biol 217) is taught. Speakers are students registered in the class, who throughout the quarter have written a review paper on molecular biology-related topics.

In this fourth edition we had two guest speakers, who took the class in the past and presented at the symposium in earlier editions.

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Wednesday, February 17

GUEST SPEAKERS

8:00-8:30	Analysis of branching in the lycophyte genus Selaginella Eric R. Schultz
8:30-9:00	Engineering increased oil production in non-seed tissues of rutabaga (Brassica napobrassica) Alan Bowsher

SYMPOSIUM TALKS

9:00 - 9:15	Prion diseases Melissa Straub
9:15 - 9:30	DNA and its forensic use in cold cases Rachel Butvin

Thursday, February 18

8:00 - 8:15	Chimerism and its consequences on the Innocence Project Jake Lewis
8:15 - 8:30	Nanoparticles: Novel approach for the battle against cancer Will Proctor
8:30 - 8:45	Gene Therapy: A potential Cure for Cancer Brooke Fleming
8:45 - 9:00	From RFLPs to STRs: The Historical Journey of DNA profiling in Forensic Science Katelyn Avendt
9:00 - 9:15	Genetic Basis for Homosexuality in Males Sonia Dhaliwal
9:15 - 9:30	The use of CODIS in DNA Profiling, and its future prospects Lindsey Pruneski
9:30 - 9:45	Parkinson's Disease and Gene Therapy Shannon Bruewer

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Lecture - Molecular techniques

Two-dimensional polyacrylamide gel electrophoresis from E. coli protein extracts
(From Alberts et al. 2008. Molecular Biology of the Cell, 5th edition. © Garland Science)

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Today we started discussing the topic of molecular techniques. There is no single chapter associated in the textbook. The material is a summary taken from miscellaneous sources.

We covered the basics on techniques on protein analysis. We focused on column chromatography (HIC, SEC, Ion-exchange, and affinity), two-dimensional gel electrophoresis and western blotting.

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Lecture, chapter 12 - RNA processing

Today we finished chapter 12, on RNA processing.

We discussed the process of intron splicing, in which snRNPs ("snurps") play an important role, the different mechanisms of alternative splicing (promoter selection, tail site selection, exon cassette selection, trans-splicing), base modification (methylation and pseudo-urydilation), RNA editing, export of RNA to the cytoplasm, and mRNA degradation (in prokaryotes and eukaryotes).

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Lab 14a - Bionformatics

Screenshot of a typical BLAST output
(click on pic for a full size image)
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Today we had an extremely shallow introduction to the universe of bioinformatics. We had an overview of the main genetic information repositories in the U.S., Europe, and Japan (see previous post), with emphasis on the NCBI website, specially on its main database, GenBank, and one of its main tools, BLAST.

The goal for this lab is for students to get acquainted with BLAST, by "blasting" nucleic acid and protein sequences (the verb 'to blast' makes reference to using the BLAST feature in the NCBI website).

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Bioinformatics links for lab 14

The following links will provide fast access to the web pages you need to do your bionformatic exercises:

• National Center for Biotechnology Information (NCBI)
• DNA Data Bank of Japan (DDBJ)
• European Bioinformatics Institute (EBI)

• GenBank statistics
• Clustal W
• Phylogeny software

• NCBI's Coffee Break

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Lecture, chapter 12 - RNA processing

Today we started covering chapter 12, on RNA processing.

We discussed the kinds of processing that different RNA molecules undergo, in both eukaryotes and prokaryotes. We compared the kinds of RNA found in bacteria (regulatory RNA and tmRNA) with those find in eukaryotes (snRNA, snoRNA, scRNA, miRNA, siRNA...), of course, besides the "classics": tRNA, rRNA and mRNA, present in all cells.

We discussed mRNA processing more in depth than in previous chapters, and we introduced the basics of rRNA and tRNA post-transcriptional processing. When focused on mRNA we talked about 5'-capping and polyadenylation ("tailing"). On Friday, we'll talk about intron splicing.

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Exam 2

Today we had our second exam.

Statistics:

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Lab 12 - Polyacrylamide Gel Electrophoreses (PAGEs) of GFP

Today we discussed a new kind of electrophoresis: Polyacrylamide Gel Electrophoresis (PAGE) is a process that uses the same principle of agarose gel electrophoresis, but it uses a polyacrylamide gel, a thinner, more expensive kind of gel that provides a higher resolution than its agarose counterpart.

Specifically we were supposed to use it to run protein samples and in two ways.

• A native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge.
• A denaturing gel, in which the proteins are denatured with high temperature and kept denatured by SDS contained in the electrophoresis buffer, so their rate of migration through the gel depends exclusively on size.

The goal was to estimate the size of the green fluorescent protein (GFP) by comparing its migration through each gel with the migration of a molecular weight ruler (a "protein ladder") loaded onto the same gel. Unfortunately we didn't have the reagent to stain the gels (coomasie), so we didn't actually run the gels, although we did talk about the process.

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Lab 11 - Protein quantitation

Wednesday, February 3, 2010

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Bradford reagent, based on Brilliant Blue G-250) to bind to proteins (causing the dye reagent to change to a different color) and measure its absorbance. The more concentrated the protein it binds, the darker the blue resultant color, and the greater the absorbance at 595 nm.

Two relative standard proteins are used, bovine serum albumin (BSA)and bovine gamma-globulin (BGG), to generate absorbance vs. protein concentration curves and then interpolate the absorbance of problem samples to estimate their concentration. The protein samples obtained from the Hydrophobic Interaction Chromatography (HIC) are used as problem samples.

This method is applied when researchers in proteomics discover a new protein and are trying to gather information about it. In our case, we "discovered" GFP, although we wouldn't have a name yet, had it been a truly newly discovered protein.

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Lecture, Chapter - Gene regulation at the RNA level

Today we covered chapter 11, on gene regulation at the RNA level.

We discussed mechanisms of gene regulation between transcription and translation:

• Rate of degradation of mRNA
• Modification of mRNA
• Control of translation by RNA binding proteins
• Anti-sense RNA
• Ribosome alteration (preferential translation of mRNAs)
• RNA interference (RNAi)
• Micro RNA (miRNA)
• Riboswitches

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