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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