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)
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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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