Lecture, chapter 5 - DNA replication

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Today we finished chapter 5, on DNA replication. We went into details of how the lagging strand is synthesized, forming the Okazaki fragments, thanks to DNA looping, which allows for it's synthesis even though the helicase and polymerase III-dimer complex travels in the same direction of the replication fork. We added how the RNA primers are degraded and the Okazaki fragments are joined, by the action of ribonuclease H, DNA polymerase I, and DNA ligase.

We then discussed how DNA transcription proceeds in eukaryotes, what's the role of telomerase in reparing telomeres after each round of replication, and the differences between the action of polymerases in prokaryotes and eukaryotes.

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Module 2, Lab 06a - Nested PCR of the GAPC gene of Arabidopsis thaliana

Arabidopsis thaliana

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Today we started the exercise in which students will learn the basics of a nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in 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 after Christmas break we will run the second round, with the nested primers.

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Module 1, Lab 05 - Size Exclusion Chromatography (SEC)

Today we did the agarose gel electrophoresis for lab 04 (detection of genetic modification in crops), using 2% agarose gels.

Then we did lab 05, size exclusion chromatography (SEC), in which a sample mix of hemoglobin and vitamin B12 were separated by size by column chromatography.

Once the procedure was finished we discussed agarose gel interpretation, specifically for labs 02 and 03.

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Lecture, chapter 5 - DNA replication

Monday, December 14, 2009

Today we started chapter 5, on DNA replication.

We discussed some of the generalities of how DNA is duplicated in a cell, and introduced the concept of replication fork. We are listing the enzymes involved in the replisome as we mention each one of their roles in the process.

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Lecture, chapter 4 - Genes, genomes, and DNA

Today we finished chapter 4, on genes, genomes and DNA.

We discussed the ways in which DNA is supercoiled, both in prokaryotes and eukaryotes, similarities and differences between both, and the implications for the functioning of the cell (in terms of replication and gene expression).

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Module 1, Lab 04 - Detection of GM in crops

In this lab we will test corn and soy samples students collected in the Fall to see if they have been genetically modified (if they are Genetically Modified Organisms or GMOs). Our tool of choice for this test will be PCR.

We extracted DNA from corn and soy leaves, as well as from a certified non-GMO seed provided by Bio-Rad. We set up PCRs using primers that will amplify de 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, which are present in about 85% of all modified crops in the U.S. As a positive control for the presence of DNA, we also used primers that amplify the photosystem II chloroplast gene, which should be present in all plants, regardless of genetic modification.

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Module 1, Lab 03 - PCR of the PV92 Alu insertion locus

Wednesday, December 09, 2009

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 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 insertion, lane 2 to an individual with insertion in both chromosomes, and lane 3 to an individual with an insertion in one chromosome.

What is your genotype like?

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Lecture, chapter 4 - Genes, genomes and DNA

Today we started chapter 4, on genes, genomes and DNA, a discussion on how DNA is organized and how such organization, contrasting prokaryotes and eukaryotes, affects DNA function and replication.

We discussed the different kinds of non-coding DNA (e.g. LINEs, SINEs, introns, satellite DNA, VNTRs...) a few reasons that explain its existence, and a few applications by using them as molecular markers.

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Lecture, chapter 3 - DNA, RNA and Proteins

Today we covered most of chapter 3, on the (very) basic structure and function of DNA, RNA, and Proteins.

Me discussed important concepts like the central dogma of molecular biology, some of the properties that make DNA a key molecule for life (antiparallelism, complementarity), and some of the roles that RNA has, beyond the transfer of information from the nucleus to the cytoplasm.

In our next meeting we'll finish chapter 3, with a basic discussion about protein structure and function.

Reminder: We are meeting tomorrow, Tuesday, in Meyer 128, at 8:00 am, to make up for the class that was cancelled on Monday Nov 30th.

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Lecture, Chapter 3 - DNA, RNA, and Proteins

Today we had our first lecture of the quarter. We went over the syllabus, including a preliminary discussion about the review paper and associated presentation that each student must develop.

We covered most of chapter 3, on DNA, RNA, and proteins. We discussed about the most basic structure of nucleic acids and some of the differences between DNA and RNA.

Reminder: We will have lecture on Tuesday Dec 8 at 8:00 a.m. to make up for the lecture we didn't have on Monday Nov 30.

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Final exam

Monday, November 16, 2009

Today we had our third and final exam. Here are the stats:

(click pic above to see full-size image)

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Lab 15 - More bioinformatics

Today we "played" with the DNA sequencing module of the Bio Rad Biotechnology Explorer series. We used the electropherograms we obtained from the sequencing facility (after sending our GAPC gene sequencing reactions from lab 09) and performed some basic tasks (despite the apparent complexity of the process): Analysis of sequences (quality of data), and blasting of the sequences obtained.

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Lecture, chapters 20-25 - Molecular Techniques

Tuesday Nov 11 and Wednesday Nov 12 2009

We covered the highlights of chapters 20-25, on molecular techniques. We divided the topic into analyses of proteins and analyses of nucleic acid sequences.

We covered techniques from column chromatography and two-dimensional electrophoresis to Southern (and northern, western, and southwestern) blotting and DNA sequencing.

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Lab 15 - Bioinformatics

Today we had a very quick and shallow introduction to the field of bioinformatics.

We took a quick tour through the NCBI website, home of GenBank (a database) and BLAST (an algorithm to do sequence pairwise alignments). We started with some problem sequences and we found out what organism they came from and what DNA fragment they corresponded to.

We also introduced the Fasta file format and put together a file to do a multiple sequence alignment using ClustalW.

Remember, this was a very shallow introduction to bioinformatics. There are many more possibilities and applications that you will explore on your own, or as part of research projects when getting a job or attending grad school.

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Links for Bioinformatics lab

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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Lab 03 - PCR of the PV92 Alu insertion locus (gel electrophoresis)Lab 14 - Size exclusion chromatography (SEC)

Today we ran a gel to confirm the results of the PCR in which the target was the PV92 Alu insertion locus in our own DNA. Results will be published soon...

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Students also did lab 14, size exclusion chromatography (SEC). It is the second kind of chromatography that we have performed (the first one was hydrophobic interaction chromatography HIC)).

The kit provided a mix of hemoglobin and Vitamin B12, proteins that have very different sizes and can be easily separated using the featured technique.

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Lecture, chapter 11 - Regulation of gene expression at the RNA level

Today we finished chapter 11 on regulation of gene expression at the RNA level.

We talked about how cells have incorporated RNAi as another tool to control gene expression. We also talked about some particularities of RNA, used for control of gene expression, like riboswitches.

Next week: The main highlights of RNA processing and molecular techniques.

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Lecture, chapter 11 - Regulation of gene expression at the RNA level

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

We talked about the different ways in which an mRNA transcript may be manipulated or modified as to stop, slow, or enhance its translation. The last topic we covered was RNA interference (RNAi).

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Lab 03 - DNA extraction and PCR of the PV92 Alu insertion locus

Lab 03 had been cancelled due to the power outage and building evacuation that we had on Thursday of week 2. It was meant to be the lab to introduce students to the Polymerase Chain Reaction (PCR). We did it today.

We extracted DNA from our cheek cells and used it to set up basic PCRs.

Our target in the PCR 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, and most likely will use them for a short population genetics exercise.

The following picture illustrates the possible outcomes of our PCRs:

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

What is your genotype like?

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Lab 12 - pGLO Small Scale Plasmid Purification Lab 13 - pGLO Restriction Enzyme Digestion (RED)

pGLO plasmid and restriction map
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Today we used the bacteria we transformed with the pGLO plasmid and cloned a few weeks ago to perform a small scale plasmid DNA purification (minipreps) and isolate the pGLO plasmid again.

Then we performed a restriction enzyme digestion, RED, using the restriction enzymes EcoRI and HindIII (see restriction map).

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Exam 2

Today we had our second partial exam.

Stats:

(click image for full size view)

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Lecture, chapter 10 - Transcriptional gene regulation in eukaryotes

We finished covering chapter 10, on transcriptional gene regulation in eukaryotes. We devoted special attention to gene silencing via formation of heterochromatin, a process in which histone acetylation and cytosine methylation are key processes.

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Lab 11 - Polyacrylamide Gel Electrophoreses (PAGEs)

Friday, October 23, 2009

Today we did a new kind of electrophoresis, or you could say that we did two. 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 used it to run protein samples and we did it in two ways. We ran a native gel, in which the proteins migrate at different rates depending on their size (molecular weight), tertiary structure, and charge. We also ran 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.

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Lab 10 - Protein Quantitation (Bradford Protein Assay)

From the Quick Start™ Bradford Protein Assay

instruction manual (BioRad)

Today we did a protein quantitation using BioRad's Quick Start™ Bradford Protein Assay, a method in which a dye reagent is used (Coomassie Brilliant Blue) to bind to proteins 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 standard proteins are used, bovine serum albumin (BSA) and gamma-globulin, 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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Lectures, chapters 9 & 10 - Transcriptional gene regulation in prokaryotes and in eukaryotes

Yesterday we covered most of the relevant section in chapter 9, on transcriptional gene regulation in prokaryotes. Today we finished and started chapter 10, on transcriptional gene regulation in eukaryotes.

Transcriptional gene regulation in prokaryotes covers the basics of gene regulation, including the concepts of global and specific regulation, activators and repressors, and regulation at transcription termination.

Transcription gene regulation in eukaryotes adds layers of complexity, like the role played by specific transcription factors, accessibility to DNA condensed in nucleosomes, and the role played by a mediator complex during gene activation.

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Lab 09 - DNA sequencing

Friday Oct 16 2009

Today we ran gel electrophoreses to confirm if the plasmid extraction (lab 8) was successful and if we had the GAPC gene (from Arabidopsis) insert. In some cases we did.

Using the samples that had the insert we mixed the miniprep 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

The report from lab 9 will be merged with the report of lab 15 (Bioinformatics)

While the gels were running we discussed 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 sequencing process.

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Lab 08 - Ligation & Transformation

Today we almost finished lab 08, the ligation and transformation module (ligation of the Arabidopsis GAPC gene into the pJet1.2 plasmid; transformation of E. coli).

We did a plasmid DNA extraction (minipreps) from the bacterial cultures we did last week, and initiated a restriction enzyme digestion with BglII to confirm if we have plasmid DNA and the insert we are interested in. Tomorrow we will run an agarose gel electrophoresis to confirm the results.

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Lecture, chapters 7 and 9 - Protein structure and function & Gene regulation in prokaryotes

Today we finished chapter 7, on protein structure and function. We discussed the main structural motifs found in DNA-binding proteins, and talked about protein denaturation.

We also started covering chapter 9, on gene regulation on prokaryotes, focusing on regulation at the transcription level. We discussed the importance of gene regulation and some of the key players involved in the process.

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Lecture, chapter 7 - Protein structure and function

Today we covered most of chapter 7, on protein structure and function. We further discussed the secondary structure of proteins (α-helices and β-sheets), and we talked about the tertiary and quaternary structures as well.

In terms of function we mainly discussed how DNA-binding proteins can read the information in the double helix without breaking the hydrogen bonds between bases.

Reminder: The first draft of the review paper is due tomorrow at noon. Send me an electronic file (preferably a Word document) before then.

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Labs 07 and 08

Friday October 09 2009

Lab 08 - Ligation and transformation

Students used bacterial colonies from the LB/Ampicillin agar plates incubated yesterday to inoculate LB/Ampicillin broth media. The broth media will be incubated for approx. 24 hours at 37ºC in a shaking water bath at approx. 200 rpm.

Lab 07 - Protein purification by chromatography

Students used the bacterial cultures inoculated yesterday to isolate Green Fluorescent Protein (GFP) by the process of Hydrophobic Interaction Chromatography (HIC).

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Labs 06, 07, and 08

Thursday October 08 2009

Lab 06 - pGLO bacterial transformation

Students analyzed the results of the bacterial cultures in LB, LB/Ampicillin, and LB/Ampicillin/Arabinose agar plates. A colony in the latter will be used to start lab 07.

Lab 07 - Protein purification by chromatography

Students used a colony from the LB/Ampicillin/Arabinose agar plates generated in lab 06 to inoculate an LB/Ampicillin/Arabinose broth tube. The broth was incubated at 37ºC for approx. 24 hours in a shaking water bath at approx. 200 rpm.

Lab 08 - Ligation and genetic transformation

Students prepared competent cells (E. coli) and transformed them with the pJet1.2 plasmid that has the GAPC gene (from Arabidopsis) insert.

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Lecture, chapter 7 - Proteins

We finished chapter 6, on transcription of genes, and went ahead and started chapter 7, on protein structure and function.

We talked about amino acids, the monomers that make up polypeptides, and how they are organized in four levels of structure. We also made the distinction between a polypeptide and a protein. We took a closer look to the primary and secondary structures of polypeptides (and proteins).

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Lecture, chapter 6 - Transcription of genes

Today we covered most of chapter 6, on transcription of genes.

We compared some details of transcription in prokaryotes and eukaryotes, including the kind of RNA polymerases involved in each one, the transcription factors involved in eukaryotic transcription, and some of the components of regulatory DNA.

Reminder: Next Wednesday (Oct 14) is the due date for the first draft of the review paper. The more advanced the paper the more feedback I'll be able to give you!

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Lab 06 - Bacterial genetic transformation with pGLO Lab 08 - Ligation and transformation (gene GAPC)

We performed the genetic transformation of E. coli cultures with BioRad's pGLO™ plasmid, engineered to contain the green fluorescent protein (GFP) gene , originally isolated from the crystal jelly Aequorea victoria.

We also performed the ligation of our GAPC gene (obtained in lab 05) with BioRad's pJet1.2 plasmid. In the next lab we will genetically transform bacteria, just as we did earlier today.

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Lab 05 - GAPDH Nested PCR

Thursday, October 1st 2009

We ran the gels for the nested PCRs we performed last week.

Due to failure of starter bacterial cultures we couldn't start lab 6, on bacterial genetic transformation, but we did talk about the main topics the lab is related to: Gene regulation, antibiotic resistance, and genetic transformation.

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Exam 1

Today we had our first partial exam.

Results and stats:

(click image for full size view)

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Lecture, Chapter 6 - Transcription

After finishing chapter 5, on the few details that differ in eukaryotic DNA replication compared to that in prokaryotes, we started chapter 6, on transcription.

We talked about the basics of the synthesis of transcripts, and got acquainted with some new terms like cistron, open reading frame (ORF), monocistronic and polycistronic mRNAs, and operon.

Tomorrow: Exam 1...!!!

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Lab 05 - GAPDH Nested PCR

Arabidopsis thaliana

_______________________________________________

Today we started the exercise in which students will learn the basics of a nested PCR. We will work with the gene that encodes one of the GAPDH isomers, GAPC, in 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, Friday, we will run the second run, with the nested primers.

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Lecture, Chapter 5 - DNA replication

We continued covering chapter 5, on DNA replication. We finished talking about DNA replication in prokaryotes, topic in which students should have an understanding of the replication fork, including the functioning of all the enzymes involved in the process.

We started talking about DNA replication in eukaryotes, organisms in which some differences are found, mainly because of the linearity of chromosomes. In prokaryotes chromosomes are circular.

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Lecture, Chapters 4 and 5 - Genes, Genomes, and DNA & DNA Replication

Today we covered the end of chapter 4, focusing mainly on the mechanisms prokaryotes and eukaryotes use to supercoil their DNA.

Then we started with chapter 5, on DNA replication. We introduced the concept of replication fork and went over some of the issues the cell has to solve in order to get supercoiled DNA to replicate.

Reminder: We are in week 3 and students should be meeting with me to decide the topic of the review paper.

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Lab 04 - Detection of genetic modification in crops

Friday Sep 18 2009

We started the exercise in which we will test corn and soy samples students brought to the lab to see if they have been genetically modified (if they are Genetically Modified Organisms or GMOs).

We extracted DNA from corn and soy leaves, as well as from a certified non-GMO seed provided by Bio-Rad with the kit. We set up PCRs using primers that will amplify de 35S promoter of the cauliflower mosaic virus (CaMV 35S) and the nopaline synthase (NOS) terminator of Agrobacterium tumefaciens, which are present in about 85% of all modified crops in the U.S. As a positive control for the presence of DNA, we also used primers that amplify the photosystem II chloroplast gene, which should be present in all plants, regardless of genetic modification.

Next week we will run an agarose gel electrophoresis to confirm the results.

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Lab 03 - PV92 PCR (postponed)

Thursday Sep 17 2009

Due to the power outage in Ada and subsequent evacuation of Meyer Hall and the Mathile Center, the lab has been postponed until weeks 09 and 10. That way we will be able to still do the lab without disrupting the flow of the following labs, which sequence is of greater importance than that of the PV92 PCR lab.

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Lecture, Chapter 4 - Genes, genomes, and DNA

Today we finished covering chapter 3, on nucleic acids and proteins, and started covering chapter 4, on genes, genomes, and DNA (2nd stop on the 'roadmap': How DNA is organized in organisms, and how such organization affect its function).

We also had our first quiz, and I announced that tomorrow we are having another one, so we can keep with the average of 1 quiz/week.

Reminder: List of topics for the review paper is due next week. Each team (of 2 people) should make an appointment with me to go over the list, and pick the topic.

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Labs 01 and 02

Thursday Sep 10

Using the micropipette

On Thursday students got acquainted with their new best friend, the micropipette. We did a couple of exercises to make sure measurements were made accurately and that students learned how to use a combination of micropipettes to measure different values. The report was handed in at the end of the lab.

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Friday Sep 11

Restriction Enzyme Digestion (RED) and analysis of lambda DNA through gel electrophoresis

In this lab we used three different restriction enzymes EcoRI, PstI, and HindIII to digest (cleave) DNA from the lambda bacteriophage. 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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Lecture, Chapter 3 - DNA, RNA, and Protein

Today we had our first lecture, and we covered most of chapter 3, on the structure and basic function of DNA, RNA, and proteins.

We covered the basic structure of nucleic acids and some of the main differences between DNA and RNA. We also mentioned a few key characteristics of the structure of chromosomes.

Next week we'll finish chapter 3 and start chapter 4, on genes, genomes and DNA ("how DNA is organized in organisms" according to the road map [see Power Point presentation from first meeting]).

Reminders:

• Two quizzes next week
• Topics of choice for review paper in two weeks

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Fall 2009...!!!

Welcome to the Fall 2009 version of Introduction to Molecular Biology...!!!

In today's session we went over the syllabus and explained some of the main components of the class. Besides the syllabus hard copy you have by now you can download it from the class WebCT site as well as from the p-drive (under 'a-cordoba').

Reminders:

• You must read chapters 1 and 2 in the textbook, or be comfortable with the material. We will not lecture on those chapters but the information is important as background for the rest of the course.
• Pair up with a classmate in order to prepare for writing the review paper in a topic on molecular biology. As of now there are 22 registered students in the class, so there is no need for a team of three. Choose wisely, since part of your grade will depend on your team-mate (see syllabus).
• Prepare a list of three topics you would like to develop in the review paper and the reasons for choosing them. By week 3 you must meet with me, out of class, so we can choose one of the topics, based on your reasons, relevance for your future career, and relevance for the class.

Any ideas to improve this blog will be greatly appreciated. What would you like to see posted here?

Have a great quarter!

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Links for Lab 15 (Bioinformatics)

• 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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Lectures - DNA repair + Control of gene expression

Between last monday and today we have finished the section on DNA repair and have covered most of the section on control of gene expression.

Last Monday (Jan 26) we closed the DNA repair section, covering the concept of emergency DNA repair: When extensive damage in the DNA is detected by RNA polymerase and is repaired not just by the regular DNA polymerase, but also by a battery of DNA polymerases that are less accurate, but more specific for a type of damage. They also lack proofrweading capacity, so the likelihood that there will be mistakes during the repair process is higher than with other DNA repair mechanisms.

Between Friday (Jan 30) and today, we have been studying the basics of control of gene expression. So far we have focused on transcriptional control, and on Monday we will focus on mechanisms of post-transcriptional control.

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Lab 12 - Small-scale plasmid DNA purification (minipreps)

Thursday Jan 29 2009

We went back to the bacterial cultures we had of transformed bacteria (E. coli), to reverse (in a way) the process we started. In this case we want to isolate the plasmid (Bio-Rad's pGLO) that we used to genetically transform the bacteria. In the process of cloning DNA this is one of the steps you follow to study the DNA segement of interest, in our case the GFP gene contained in the pGLO plasmid. We made zillions of copies of it, and now we have to extract it from the bacteria to analyze it.

We used Promega's Wizard® Plus SV minipreps DNA purification system. An easy to use kit to purify plasmid DNA in a lab like the one we have available.

Next week we'll run a confirmation gel and perfomr a restriction enzyme digestion (RED) of the pGLO plasmid.
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Lab 11 - Polyacrylamide Gel Electrophoresis (PAGE) of GFP samples

Thursday Jan 29 2009

This lab should have taken place on Wednesday Jan 28, but classes were cancelled due to weather.

Polyacrylamide Gel Electrophoresis (PAGE) is a technique used for separating polynucleotides or polypeptides that are very similar in size, providing greater resolution than with an agarose gel.

We used PAGE to measure the size of a protein, GFP. If we follow the series of "make believe" in which we are dealing with a new protein, this would be a step to find more information about it. Protein size is quantified in Daltons (Da), a measure of molecular mass. One Dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10-24grams (g).

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 ran two types of PAGEs: Native and denaturing (a.k.a. SDS)

Proteins can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all. In native gel electrophoresis the proteins being separated differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Because of different charges and tertiary structure proteins of the same mass may migrate at different rates.

In SDS gel electrophoresis 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 all the proteins have 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 is relative only to its size (molecular weight) and not its charge or shape.

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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Lecture - DNA repair

Yesterday we covered most of the DNA repair section in chapter 5.

We talked about the most common spontaneous DNA changes (e.g. depurination and deamination), the importance of having genetic information coded in a double helix, the main mechanisms of DNA repair (base excision repair, and nucleotide excision repair), and transcription-coupled repair.

On Monday we'll talk about emergency DNA repair and repairing double strand breakes.

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