Lecture, chapter 5 - DNA replication

We finished the chapter on DNA replication.

We discussed the process in which the DNA polymerase complex actually replicates DNA, including the DNA looping that allows both strands to be synthesized at the same time.

We also covered eukaryotic DNA replication, emphasizing the differences with prokaryotes.

Watch the following video or access this link to understand the main features of the replication process

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Section 1Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Same lab as with section 2. Click here for information on it.

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Lecture, chapter 5 - DNA replicaiton

Today we started chapter 5, on DNA replication, and the third stop in our roadmap, on how genetic information is preserved and inherited.

We started a discussion on how DNA is replicated in prokaryotes, including the concepts of replication fork and replisome. We described the role of several enzymes on the process of DNA replication (DNA gyrase, DNA helicase, single strand binding (SSB) proteins, primase, DNA polymerase), and the fact that there are a leading and a lagging strand during the replication process, the latter being extended in bursts forming Okazaki fragments.

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Section 2Module 2, Lab 05 - GAPDH nested PCRAmplifying the GAPC gene

Thale cress, Arabidopsis thaliana
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Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and on Wednesday we will do the second run, with the nested primers.

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Section 2Module 1, lab 4 - PCR of the PV92 Alu insertion locus

Today section 2 completed lab 4 in module 1. For more information click on PCR of the PV92 Alu insertion locus

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Lecture, chapter 4 - Genes, genomes, and DNA

Organization of the human genome
from Allison, L. 2007. Fundamental Molecular Biology. Blackwell Publishing.
(click on pic for a full size image)
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Today we followed our discussion on how DNA is organized in genomes (second stop in our roadmap), including a discussion on satellite DNA (satellites, minisatellites, and microsatellites), palindromic DNA (mirror-like palindromes, inverted repeats, hairpins, stem-and-loops), junk and selfish DNA, and supercoiling.

On Monday we'll discuss how eukaryotic DNA is compacted enough to fit the nucleus of a cell

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Module 1 (section 1)Lab 3 - PCI DNA extraction from human bloodLab 4 - PCR of the PV92 Alu insertion locus

Lab 3 - PCI DNA extraction from human blood

Yesterday students finished the PCI DNA extraction from their own blood. The steps that were left included adding the PCI, doing some pellet washes with cold ethanol and eluting the DNA in TE buffer. Samples were incubated overnight at 55ºC.

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Lab 4 - PCR of the PV92 Alu insertion locus

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

Our target is the PV92 Alu insertion locus, located on chromosome 16.
Alu elements are a family of short interspersed repetitive elements (SINEs) that have mobilized throughout primate genomes for the last 65 My, by retrotransposition.

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 PV92. Because the PV92 insertion locus 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.

In this lab we will test the presence of 0, 1, or 2 PV92 Alu insertions in our genomes.

The following picture illustrates the possible outcomes of our PCRs:

The sample on lane 1 belongs to an individual with no PV92 Alu insertions, 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?

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Module 1, lab 3 (section 1) - PCI DNA extraction from human blood

Today we started the phenol-chloroform isoamyl alcohol (PCI) DNA extraction from most students' blood. Blood samples were obtained throughout Monday and Tuesday.

Students added SSC buffer (pH stabilization), SDS (cell lysis), sodium acetate (NaOAc; protein precipitation), proteinase K (inactivation of endonucleases), PCI (separation of proteins and nucleic acids), and 100% ethanol (DNA precipitation). Samples were frozen to continue the process tomorrow.

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LectureChapter 3 - DNA, RNA, and ProteinsChapter 4 - Genes, genomes, and DNA

Today we finished chapter 3, mentioning the basics of the different functions of RNA and protein structure.

We then started chapter 4, on genes, genomes and DNA, which is our second strop in the class roadmap: How DNA is organized in organisms and how such organization affects its function.

We discussed how little genetic information is necessary for independent life and the importance of non-coding DNA in eukaryotes. We talked about pseudogenes, introns, and repeated sequences (tandem repeats and intersperse elements).

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Module 1, lab 3 (section 2) - PCI DNA extraction from human blood

Students extracted DNA from their own blood. The entire protocol was completed and samples of DNA eluted in TE buffer were frozen at -20ºC.

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Module 1, lab 2 (section 2) - Size exclusion chromatography

Students did a size exclusion chromatography of a protein sample containing hemoglobin and vitamin B12. For more a more detailed blog entry click here.

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Module 1, lab 1 (section 2) - RED of lambda DNAGel electrophoresis

Students completed ran an agarose gel electrophoresis of the restriction enzyme digestion performed last week. For more details check this previous blog entry.

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A bacterium that uses arsenic (and criticisms of the original paper)

Gammaproteobacteria GFAJ-1,
a bacterium capable of using arsenic as a component of its cell machinery (photo: NASA Astrobiology)

Left: Felisa Wolf-Simon, NASA astrobiology research fellow, processing
mud samples at Mono Lake. Right: Mono Lake, California (photos: NASA Astrobiology)
_________________________________________________

Yesterday NASA made an exciting announcement in biology:

"Researchers conducting tests in the harsh environment of Mono Lake in California have discovered the first known microorganism on Earth able to thrive and reproduce using the toxic chemical arsenic. The microorganism substitutes arsenic for phosphorus in its cell components."

(From NASA Astrobiology)

This is a major finding, with important implications in the fields of astrobiology, microbiology and molecular biology, since P is one of the six elements so far believed to be essential to every life form. The Gammaproteobacteria GFAJ-1 is the first exception ever found to that rule. It was found in Mono Lake, California.

The discovery was published on the on line version of Science Magazine, and will soon be published on the regular paper edition. Pdfs of the article and support material can be accessed in my p-drive (a-cordoba. ONU students and faculty only).

In the following weeks, as we learn more about this discovery I'll bring new information into the classroom. In the meantime I want to provide links for you to start your own exploration of the topic:

• NASA announcement
• NASA Astrobiology
• National Public Radio (NPR)
• Wired Science
• CNN
• Discover Magazine
• Popular Science

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Update, March 21 2011

The news is exciting indeed, but there are detractors. Here I give them a voice (more updates will come if I find the sources)

Felisa Wolfe-Simon (of arsenic infamy) is no more convincing in person than in print
By Michael Eisen | March 18, 2011

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Update, June 01 2011

Science Magazine will publish a paper with criticisms to Wolfe-Simon et al.'s paper.  Click here to see the press release on Science Magazine News. (Link to the Science article will follow soon)

Click here for another Science Magazine News article on the criticism's to Wolf-Simon's paper.

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Lecture, chapter 3 - DNA, RNA, and proteins

Today, on chapter 3 we discussed the basic structure of nucleic acids, from the components of a nucleotide to the specialized regions in a eukaryotic chromosome. We also mentioned the central dogma of molecular biology and mentioned the several general and special cases of flow of genetic information.

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Module 1, lab 01b (section 1) Restriction enzyme digestion of lambda DNAGel electrophoresis

Today we ran the first agarose gel electrophoresis of the quarter. Students learned, or reinforced, how to load, run, take a picture of, and interpret an agarose gel.

The samples used in the gel were from the restriction enzyme digestion (RED) students set up yesterday: Lambda DNA undigested and digested with the restriction enzymes EcoRI, PstI, and HindIII. The ladder used was lambda DNA pre-digested with HindIII.

The picture below, shows the different bands of the HindIII digestion used as DNA ladder. Notice that there are 7 bands, one more than what it is specified in the lab guide. The 7th band is so faint that it is assumed to be invisible, but it was visible in most of the gels students ran. If visible, data from the 7th band should be included in the lab report.

All sizes in bp
(click on pic for full size image)
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Module 1, lab 02 (section 1) Size exclusion chromatography (SEC)

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, labs 00 and 01 (section 1)Restriction enzyme digestion (RED) of lambda DNA

Micrograph and structure of a bacteriophage
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We started with a series of exercises to learn how to use a micropipette. Once students became familiar with the instrument we started with lab 1.

Lab 1 (module 1) - Restriction enzyme digestion (RED) of lambda DNA

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.

As a DNA marker, or DNA "ladder", we used a sample of lambda DNA pre-digested with HindIII.
Students will measure the distance bands in the gel migrated and will infer the size of the different bands based on such information.

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Biol 217, Winter 2010-11

Welcome to the Winter 2010-11 version of the Intro Molecular Biology (Biol 217) class.

Today we had our first official meeting, and due to several last minute registered students there has been a change in the meeting room. The class was originally scheduled to meet in Mathile 107, and indeed that's the room where we met today. But starting Friday we will meet in Meyer 128.

Today we reviewed the syllabus, explaining the grading scheme, some assignments (literature review paper and symposium presentation), and expectations in the class. We also went over the rationale of the class and how it explains the sequence of lectures that will be taught.

Reminders:

• Fall 2010 power point presentations are available on WebCT and the p-drive (under a-cordoba)
• This quarter's power point presentations will be made available as lectures are taught
• This blog can be used as a reference of the class progress; check it often, specially if you have missed class

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Module 1, labs 00 and 01 (section 2)Restriction enzyme digestion (RED) of lambda DNA

Today we had the firs lab meeting with section 2 in the class.

We went over the lab syllabus, distributed materials (lab notebooks, lab coats, and permanent markers), introduced the lab routines (where to find materials and how to behave in the lab), and spent a fair amount of time in the proper use of micropipettes.

We then performed lab 00, which allows students to practice pipetting techniques, and then we started lab 01, a restriction enzyme digestion.

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Exam 3 - Final

Stats :

Click on pic for a full size image

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5th ONU Intro Molecular Biology Symposium

Department of Biological and
Allied Health Sciences
Mathile Center 107
November 11 - 12, 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.

Click on pic for a full size image

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Module 4, Lab 14 - Bioinformatics (Phylogeny)

When DNA samples are sent to a sequencing facility the return are files with information. The specific files that a researcher receives are called electropherograms ("recordings of the separated components of mixtures produced by electrophoresis”).

We did a basic analysis of some electropherograms of the Ribonucleotide Reductase Small Subunit (RRss) gene from animals of several phyla. We saved the information in fasta format and did a multiple sequence alignment using ClustalW. We generated a nexus file which was finally used to do crude phylogenetic analyses using the software package PHYLIP on the web.

The exercise was just an example of one of the many possible sequences of steps that can be followed to analyze genetic information. The main point was to go from electropherograms to analysis, even though the ways to analyze DNA sequence data are far too many to cover in a single lab.

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Lecture - Molecular techniques (nucleic acids)

In todays lecture we focused on techniques that are used for analyzing nucleic acids. I decided to focus mainly on DNA sequencing, given how important that such technique has become in the last decade. Topics that were discussed:

• PCR
• PCR in disease diagnosis
• DNA sequencing
• Chain termination method (Sanger method - manual and automated)
• Shotgun sequencing
• Pyrosequencing
• Next-generation sequencing
• DNA typing

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Lecture - Molecular techniques (proteins)

In this lecture we had a snapshot of what could easily be a whole course just on molecular techniques. We focused on protein analysis techniques:

• Purification (column chromatography)
• Separation (SDS PAGE and two-dimensional electrophoresis)
• Detection (western blotting)
• Predicting function (using bioinformatic tools)

We also introduced the basics of techniques focused on nucleic acids, including a discussion of the constantly decreasing cost of sequencing complete genomes.

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Module 4, Lab 13 - Links for Bioinformatics lab 1

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

• Fasta format description
• ClustalW2 (EBI)

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Module 3, Lab 12 - RED of pGLO plasmid

Today we did a restriction enzyme digestion (RED) of the plasmid DNA purifications (minipreps) we did last week. The goal was to isolate the GFP gene from the pGLO plasmid. After the cloning process that would have been a step before purifying the GFP gene sample for further study.

We used the restriction enzymes EcoRI and HindIII (individually and in combination) to reach our goal. We confirmed the results with an agarose gel electrophoresis (students in section 2 even did a "retrophoresis"... Hopefully it was a very valuable lesson)

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Lecture, chapter 11 - Gene regulation at the mRNA level

Today we covered chapter 11, on gene regulation at the mRNA level. Regulation mechanisms in the middle ground between transcriptional regulation and translational regulation.

We discussed the control of rate of degradation of mRNA, the effect of translational regulatory proteins (activators and/or repressors), regulation by anti-sense RNA, and regulation by alterations to the ribosome.

Tomorrow we will discuss RNA interference (RNAi)

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Module 3, Lab 11 - PAGEs of GFP

Image from Bio Rad

As a follow up of Thursday's lab, we stained the gels with Coomasie G-250 stain and then air dried them for analysis.

(click on pic for a full size image)

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Module 3, Lab 12 - Small Scale Plasmid DNA Purification of pGLO

In this lab students purified the pGLO plasmid following Promega's Wizard Plus SV Minipreps DNA Purification System®.

Next week we will do a restriction enzyme digestion of the plasmid DNA in order to isolate the GFP gene from the plasmid.

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Module 3, Lab 11 - Native and denaturing polyacrylamide gel electrophoreses (PAGEs) of GFP

Today we ran 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.

We specifically ran protein samples in two ways.

• A native gel, in which the proteins, in their native state, migrate at different rates depending on their size (molecular weight), 3D structure, and charge.
• A denaturing gel, in which the proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge and a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which they migrate in the gel depends only to their size (molecular weight) and not its charge or shape.
  

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.

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa.

We will be able to compare teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).

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Exam 2

Statistics for exam 2

Click on pic for full size image

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Module 3, Lab 10 - Protein quantitationBradford dtermination of GFP

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 from a reddish-brownish color to blue) 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 (mostly with GFP) to estimate their concentration. The problem samples were obtained from the Hydrophobic Interaction Chromatography (HIC).

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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Module 3, Lab 09 - Hydrophobic Interaction Chromatography (HIC) of GFP

Friday, October 21, 2010

Today we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.

A sample of bacteria was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.

A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected should glow.

Diagram of Hydrophobic Interaction Chromatography (HIC)

GFP molecules are represented by black triangles

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Module 2, Lab 07 - Sequencing reactions of GAPC gene

After the cloning process of the GAPC gene from Arabidopsis, and extracting the plasmid DNA (to isolate the pJet1.2 plasmid) we did a RED to confirm the success of the ligation.

Using the samples that had the GAPC gene insert we mixed purified plasmid DNA with forward and reverse sequencing primers (pJET SEQ F and pJET SEQ R), and put them in a 96-well plate. The plate will be shipped to the DOE Joint Genome Institute (JGI) to be sequenced as part of their Sequencing Training Program (STR). The results should be in in two weeks, ready to be used in the bioinformatics labs

We will discuss the DNA sequencing technique most commonly used: Dye-terminator sequencing, a modification of Sanger's chain termination sequencing protocol, which allowed the automation of the DNA sequencing process.

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Guest lecture by Dr. Renee Reijo-PeraEarly human embryo development and associated gene expression

We had the privilege of having Dr. Renee Reijo-Pera, the director of the Human Embryonic Stem Cell Research Center at Stanford University, as a guest lecturer in our class. She shared with us her lab's findings in recent years on stem cell research and early human embryo development.

Some of the main topics in her lecture included...

• Maternal vs. embryonic gene expression - Stages at which maternal mRNAs are active, and then degraded, and at which embryonic mRNAs are synthesized
• Dynamics of cell division between fertilization and blastocyst stage
• Prediction, at day 2 of development, of which embryos are viable (will successfully reach blastocyst stage) - Development of an algorithm to make an objective prediction
• Things we do not know about human embryo development and how stem cell research can help
• How embryo images were obtained and made into movies to allow analysis of developmental process
• Analysis of gene expression - analysis of mRNA from 96 selected genes, extracted from a single cell
• How the development process is correlated with patterns of gene expression

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Lecture, chapter 11 - RNA processing+ info about guest lecturer, Dr. Renee Reijo-Pera

Today we finished chapter 11 on RNA processing. We focused mainly on alternative splicing and how it produces different mRNA molecules by transcribing the same gene.

We also discussed processes like base modification, base substitution, RNA editing, and RNA degradation.

After finishing the chapter we discussed students' impressions on Dr. Reijo-Pera's Keiser lecture yesterday evening and expectations for her talk in our class tomorrow...!!! (expectations from the talk itself and about students' interaction with Dr. Reijo-Pera)

Tomorrow:

Dr. Renee Reijo-Pera, from Stanford University, will give a lecture on human preimplantation development and gene expression and pathways during the first few days of development. We will be joined by students in Dr. Aulthouse's Developmental Anatomy class, and potentially Dr. Walden's CLS program so the room will be packed. The talk will be as exciting as the Keiser lecture and having two-three classes in the audience will make the discussion more interesting and lively...!

Students should be ready to ask questions to, and engage in a discussion with, Dr. Reijo-Pera. Please check the following links:

• The Reijo-Pera lab
• People at the Reijo-Pera lab
• Dr. Reijo-Pera's publications
• Dr. Reijo-Pera's contact info

It will be an exciting day. Take advantage of it!

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Upcoming lecturer: Dr. Renee Reijo-Pera

On Wednesday we will have the honor of having Dr. Renne Reijo-Pera as a guest lecturer in our class. Dr. Reijo Pera is visiting ONU as the Distinguished Keiser Lecturer of 2010.

Her lecture is titled Human Health, Development and Stem Cells, and it will be delivered on Monday, October 18, at 7:00 pm at the Freed Center

In our class Dr. Reijo-Pera will be talking about human preimplantation development and gene expression and pathways during the first few days of development. This topic is highly appropriate for our class and it will provide you with the opportunity to interact in an intimate setting with a world-class researcher. Take advantage of such opportunity!

Dr. Reijo-Pera is the Director of the Human Embryonic Stem Cell Research and Education Center at Stanford University. For more information see her lab web page, which includes information about her and her group, and her publications.

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Module 2, Lab 06 - Cloning - RED of the plasmid DNApurifications

(entry in progress)

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Module 2, Lab 06 - Cloning (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Today we used the E. coli cultures you inoculated yesterday to do a small-scale plasmid DNA extraction ('miniprep') using Promega's Wizard® Plus SV Minipreps DNA Purification System.

Here's a recap of what we have done so far in the last couple of exercises:

Lab 5 - Nested PCR of the GAPC gene from Arabidopsis

Lab 6 - Cloning

• Ligation of PCR amplified GAPC gene onto the pJet1.2 plasmid
• Genetic transformation of E. coli with the pJet1.2 plasmid
• Cloning of genetically transformed E. coli
• Minipreps (purification of pJet1.2 plasmid)

Next, a restriction enzyme digestion to confirm successful plasmid DNA purification (lab 6) and setting sequencing reactions (lab 7)

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Module 3, Lab 08 - Genetic transformation of E. coli with the pGLO plasmid

Jen was kind enough to prepare new LB agar plates and redo the transformation of E. coli with the pGLO plasmid. Then she plated the transformed bacteria on the plates she prepared, some of which had ampicillin, arabinose, or both.

[Fantastic] Results are shown in the pictures below:

(entry in progress)

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Lecture, chapter 11 - RNA processing

tRNA processing

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Today we started chapter 11, on RNA processing, the fifth "stop" in our roadmap.

We mentioned the most basic processes through which RNA is modified (base modification, cleavage, and splicing) and described the more complex processes that are specific to eukaryotic RNA (5' capping and 3' polyadenylation).

We discussed in more detail the steps through which introns are spliced out the pre-mRNA molecule, and introduced the concept of alternative splicing.

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Lecture, chapter 10 - Gene regulation in eukaryotes

Today we covered chapter 10, on gene regulation in eukaryotes.

We highlighted the differences in between the regulatory processes in prokaryotes and the more complex processes in eukaryotes, mainly the facts that in eukaryotes many transcription factors are needed to aid transcription and that DNA is many times condensed in heterochromatin, and therefore unavailable for RNA polymerase and other proteins.

Among some of the processes that affect gene expression we discussed histone acetylation, a mechanism through which heterochromatin is relaxed into euchromatin, making DNA available for DNA binding proteins, and methylation, a mechanism through which genes can be repressed or silenced, in some cases because it promotes de-acetylation of histones and DNA condensation into heterochromatin.

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Module 2, Lab 06 - Ligation and transformation (GAPC gene from Arabidopsis and pJet1.2 plasmid)

Today we purified the PCR products from the nested PCR lab (GAPC gene from Arabidopsis) and used them to genetically transform E. coli.

The lab was divided in three main steps

• Ligation (of GAPC gene on to the pJet1.2 plasmid)
• Preparation of competent cells
• Genetic transformation of E. coli

We spent most of the lab manipulating bacteria to make them competent (i.e. get them ready to uptake extracellular DNA). Once this was achieved, the GAPC gene from Arabidopsis, obtained via nested PCR, was ligated to the pJet1.2 plasmid.
The plasmid was then used to genetically transform E. coli, which were spread on LB agar/Amp/IPTG plates and incubated.

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Lecture, chapter 09 - Gene regulation in prokaryotes

Today we lectured for a little over one hour, as a replacement for the lab we could not do, because of not having transformed bacteria to "play" with.

We finished chapter 9 on gene regulation in prokaryotes. We discussed the concepts of positive and negative regulation, including the role of (specific) activators and repressors, and global regulators. We studied how the Lac operon works in E. coli, and set the stage for starting with gene regulation in eukaryotes.

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Lecture, chapter 09 - Gene regulation in prokaryotes

We started the fourth stop in our "road map" (how genes are regulated).

We introduced chapter 09, on gene regulation, by highlighting the importance of the process for both eukaryotic and prokaryotic cells. We also mentioned the different steps during the process of information transfer at which regulation can take place, transcription being the most common one.

Note: Because the results of the transformation lab were negative (E. coli genetic transformation failed) we will lecture tomorrow.

• Section 01: 8:45 am
• Section 02: 10:00 am
• (Any one who wants to attend the opposite section is welcome)

Information on how to obtain the results from the transformation lab will be posted on an update in that exercise's blog entry.

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Lecture, chapter 7 - Protein structure and function

We finished chapter 7...

We discussed the features that allow certain proteins to bind to DNA and also what of their most common structural motifs are. We closed by briefly describing what protein denaturation is, how it differs from degradation, and what are the most common denaturing agents.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function.

We discussed the properties a protein has given the characteristics amino acids provide to the whole structure and how they actually affect protein function. We also listed different protein categories according to different functions they may have.

Tomorrow we will talk about how proteins can recognize DNA sequences to bind to them.

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Module 3, Lab 08 - Genetic transformation of bacteria with the pGLO plasmid

Aequorea victoria, original source of the green fluorescent protein (GFP)

___________________________________________________________________

Today we used the pGLO plasmid to genetically transform Escherichia coli.

pGLO is a plasmid that has been engineered to contain the Green Fluorescent Protein (GFP) gene, originally isolated from the jelly Aequorea victoria. GFP produces a green fluorescence when excited by blue or UV light.

In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinose operon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The regulatory sequence has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and, therefore, GFP is produced.

Another feature of the pGLO plasmid is the presence of the beta-lactamase gene, which provides resistance against the antibiotic ampicillin.

The bacteria were transformed through the heat shock technique, and then plated on LB agar plates containing:
Just LB (lysogeny broth)
LB and ampicillin
LB, ampicillin and arabinose

Plates are being incubated for 24 hours at 37ºC.

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Module 2, Lab 05 - GAPDH Nested PCR

Today we ran a gel electrophoresis to confirm the results of the second round of PCR (using the nested primers).

Ideally, we would have purified the Arabidopsis PCR product (GAPC gene; in fact, section 1 did follow the process), but all PCRs failed. WHY?

Here's what I think happened:

When I retrieved the tubes from the thermocycler they had a very small volume of liquid at the bottom of the tube. Most of the volume (pretty much all the water, not just the water you added) was condensed at the top of the tube. That means that all the reagents were desiccated and molecules couldn't interact with each other. Result: no reaction whatsoever.

Probable cause: A glitch in the thermocycler. Most thermocyclers today have heated lids, to prevent condensation of water at the top of the tube. There is evaporation, but by preventing condensation water is always being recirculated in between its liquid and gas states and there is always enough liquid water to keep the PCR going. If the lid doesn't heat up during the process, then most of the water evaporates, condensates at the top of the tube and the reaction is ruined.

I have no idea of why the lid wouldn't heat up, since it is an automatic process every time you run a program.

Solution: I am running the nested PCRs again. The first round is in the thermocycler as I type and I triple-checked to make sure the lid was hot. I will run the second round and a gel to make sure that we have product (GAPC gene). If the reaction works, in week 5, before the ligation and transformation exercise, you will have to purify the PCR product before proceeding.

Lesson: Learn how to deal with frustration. In molecular biology many things can go wrong when following a protocol and you must keep on going. If you ever become part of a research lab you will find out, first hand, that nothing is as perfect as it looks in the published literature. Today you had a little taste of it.

We must shake it off and do it again. In this case I have to do it again (but if there are any volunteers for setting up the second round of PCRs, let me know)

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Results of make up PCRs

The following gels show the results of the nested PCR (click on the pic to see a full size image):

Lanes 1 and 2 on both gels are the positive and negative controls, of the first round PCR on the left gel and of the nested PCR (2nd round) on the right. The box on the left gel shows a faint band, which resulted from leaking when I was loading the positive control in the adjacent well.

Lanes 3 and 4 on the left gel show products of the first round of PCR, and all other lanes in both gels show products of the second round of PCR.

I have saved the products of the nested PCRs for you to purify this week and go on with lab 6.

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Exam 1

Statistics of the exam:

(click on pic for full size image)

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Lecture, chapter 6 - Transcription

Today we finished the chapter on transcription.

We discussed the process in which RNA polymerase binds to the promoter in prokaryotes, generates de RNA transcript, and reaches the terminator to end transcription (Rho-independent termination and Rho-dependent termination).

We then discussed eukaryotic transcription. The promoter is more complex (it has an initiator box, a TATA box, and upstream elements), there are three different RNA polymerases that transcribe nuclear genes (mitochondrial and chloroplast genes use other polymerases), and there are proteins, called transcription factors (general and specific), that aid RNA polymerases in the transcription of genes. Some proteins may bind to enhancer regions, upstream from the promoter, to aid in the process.

Watch the video that we saw in class (embedded action disabled in Youtube)

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Module 2, Lab 05 - GAPDH Nested PCR

Thale cress, Arabidopsis thaliana

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Thursday (Round 1)

Today we started the exercise in which students will learn the basics of nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in the thale cress (Arabidopsis thaliana), the model organism of plants. Some people call it "the fruit-fly of plants".

GAPDH is an enzyme in charge of catalyzing one of the reactions in glycolysis. There are several nuclear genes that encode GAPDH isomers (proteins with different amino acid sequences but with the same function), and we are targeting the gene GAPC in the A. thaliana genome. We ran a first round of PCR, with our initial primers, and tomorrow we will run the second run, with the nested primers.

Friday (Round 2)

Today we ran the second round of PCR, using the nested primers.

In order to do so the first round primers were degraded adding an exonuclease to the amplified samples and incubating for 15 minutes at 37ºC. Then the exonuclease was denatured by incubating the samples at 80ºC for 15 minutes.

Finally, the Arabidopsis genomic DNA was diluted (1:50) and used to set up the second run of PCRs.

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Lecture, chapter 6 - Transcription

We started chapter 6 on Transcription.

We discussed general introductory issues on the process of transcription, including terminology of components and processes (e.g. template or sense strands vs. coding or anti-sense strands, hosekeeping genes, cistrons, open reading frames [ORFs], operons, and monocistronic and polycistronic DNA).

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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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