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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