Today we finished labs 3 and 4, which we started yesterday.
Lab 3 - Restriction Enzyme Digestions - Gel electrophoresis
We loaded the DNA digestions that we set up yesterday, plus a standard (a.k.a. marker or ladder). Once again, the DNA that was digested (and an un-digested control) came from the lambda bacteriophage, a common phage of E. coli. The enzymes used for the digestion were HindIII, PstI, and EcoRI. The marker was a precut lambda bacteriophage DNA, HindIII.
Methods and results at this point should resemble the ones from lab 2. You need to do a visual estiumation of size of DNA bands, and then use the precut HindIII digestion bands to create a migration distance vs. band size curve in semi-log paper and interpolate (and extrapolate) the information obtained from other samples.
Nice thing about this lab is that STUDENTS actually estimated the band migration distance, as opposed to last week, when the instrctor did it.
Lab 4 - PV92 PCR - Gel electrophoresis
Yesterday after lab, the instructor added the PCR master mix to the human DNA samples donated by the students, and put all the tubes in the thremocycler to perform a PCR to amplify the PV92 locus. Today students ran gel electrphoreses to vizualize the results and determine if they are homozygous (positive or negative) for the Alu sequence insertion in the PV92 DNA segment.
Results will be used to perform a quick and basic bioinformatics exercise using the obtained information in the Allele Server webpage. Access the Dolan DNA Learning Center Gene Almanac and click on 'Resources', then on 'Bioservers' and log on to 'Allele Server'. The username is a-cordoba, and the password is biol217.
Once in there click on 'add data'. On the pull-down menu 'please choose your group' click on 'ONU-biol 217' (which should be the default). The password is biol217 and your number is the one I assigned to you on an e-mail that I sent.
Enter the information (you don't have to enter info about your parents' descent if you don't want to) and click ok. Once all of us have done that we can play with the data and find out a few things of us as a "population".
To VIEW the information, click on 'Manage Groups', and click on 'View' in front of the 'ONU-Biol 217' group.
Click here to find more information about PV92 and the Alu sequence!
----------------
Friday, September 19, 2008
Thursday, September 18, 2008
LABS 3 and 4 - RED and PCR
Today we actually started labs 3 and 4, which will be finished tomorrow.
Lab #3
Lab 3 is very similar to Lab 2, except for the fact that this time we ACTUALLY set up the Restriction Enzyme Digestions (REDs), instead of simply running pre-cut DNA in a gel. We added DNA, restriction buffer, and restriction enzymes to a mix, and incubated it for 30 minutes at 37ºC. Why that temperature? The enzymes have been isolated from bacteria that live in mammals and, as we know, tht is our body temperature. That means that the enzymes will be most efficient at that temperature. They will still work at lower temperatures, but they would take a lot longer to do the same job. Since we incubated them at 37º, I stored them in the fridge (4º), where the enzymes won't act almost at all. If we had not incubated the digestions, they should be sitting at room temperature until tomorrow morning.
Tomorrow we will run a gel electrophoresis with such samples and compare them with the ones we ran last week in lab#2. Results should be pretty similar. Yes, you'll have to estimate the size of the DNA contained in the bands, using the visual method and the standard curve using semi-log paper. I'll e-mail you a semi-log paper pdff file in case you want to print some more copies.
Lab 4/5 - PV92 PCR
PV92 is a locus found in chromosome 16 in humans. As you should remember, we all have two copies of each chromosome, so we have two chromosomes 16, and therefore we have two copies of the PV92 locus.
What does PV92 do? Nothing! It does not code for any thing, just as the 95% of the human genome. Only about 5% of our genome actually encodes some sort of product. This locus was named in the old days of karyiotyping, when we didn't really know what different areas stained in a chromosome did. After we sequenced the human genome, we figured out that the names given to many loci didn't actually correspond to a functional gene. PV92 may be a pseudogene (a duplicated gene that lost its function), so for practical purposes we'll consider it a gene..
The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. And therefore when we think about mating in primate populations, it is random when it comes down to PV92. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population, so PV92 has been used for a lot of human population genetics studies.
There is another nice thing about PV92. Some people have an Alu sequence inserted in the very middle of the PV92 locus. This Alu insert, an example of an intron (a DNA fragment found in between coding regions of a gene, that doesn't code for any thing and that it's spliced during transcription in functional genes), makes pV92 300 bases longer, which is a difference big enough that it can be visualized in a gel (originally PV92 is about 600 base pairs long).
An Alu sequence is a retrotransposon (a DNA fragment that has been inserted in a location where it doesn't belong via reverse transcription, i.e. mRNA actually writes a sequence into the genome) found throughout primate genomes. There are thousands of copies of Alu sequences found in a single genome.
PCR, the Polymerase Chain Reaction, a method to amplify (make many copies of) specific DNA fragments, is used to generate enough DNA that it can be studied with a variety of thechniques. Using PCR we will amplify our PV92 loci to see if we have the Alu sequence insert in any of our copies of PV92. Each one of us may have 0, 1, or 2 alu inserts in our PV92 copies and we will find out through PCR and gel electrophoresis.
Today we extracted our own DNA. We scraped our cheeks to get some epithelial cells, we neutralized ions (with the InstaGel) that may be used as cofactors by DNases to chop out DNA, and then we bursted our cells open (with the water baths) to extract the DNA from the nuclei.
After lab I added your DNA and mine, plus three positive controls, to a master mix with all the constituents of a PCR: buffer, nucleotides, Taq polymerase, magnesium, and primers (synthesized single stranded oligonucleotide sequences) and put the tubes into the thermocycler. We'll talk about details of how PCR works later.
Tomorrow we'll run a gel electrophoresis to figure out who among us has the Alu insert in his/her PV92 locus, and if it is a single copy or two.
-------------
Lab #3
Lab 3 is very similar to Lab 2, except for the fact that this time we ACTUALLY set up the Restriction Enzyme Digestions (REDs), instead of simply running pre-cut DNA in a gel. We added DNA, restriction buffer, and restriction enzymes to a mix, and incubated it for 30 minutes at 37ºC. Why that temperature? The enzymes have been isolated from bacteria that live in mammals and, as we know, tht is our body temperature. That means that the enzymes will be most efficient at that temperature. They will still work at lower temperatures, but they would take a lot longer to do the same job. Since we incubated them at 37º, I stored them in the fridge (4º), where the enzymes won't act almost at all. If we had not incubated the digestions, they should be sitting at room temperature until tomorrow morning.
Tomorrow we will run a gel electrophoresis with such samples and compare them with the ones we ran last week in lab#2. Results should be pretty similar. Yes, you'll have to estimate the size of the DNA contained in the bands, using the visual method and the standard curve using semi-log paper. I'll e-mail you a semi-log paper pdff file in case you want to print some more copies.
Lab 4/5 - PV92 PCR
PV92 is a locus found in chromosome 16 in humans. As you should remember, we all have two copies of each chromosome, so we have two chromosomes 16, and therefore we have two copies of the PV92 locus.
What does PV92 do? Nothing! It does not code for any thing, just as the 95% of the human genome. Only about 5% of our genome actually encodes some sort of product. This locus was named in the old days of karyiotyping, when we didn't really know what different areas stained in a chromosome did. After we sequenced the human genome, we figured out that the names given to many loci didn't actually correspond to a functional gene. PV92 may be a pseudogene (a duplicated gene that lost its function), so for practical purposes we'll consider it a gene..
The nice thing of having a locus that doesn't do any thing at all is that it is not influenced by natural selection. And therefore when we think about mating in primate populations, it is random when it comes down to PV92. So? Well... in population genetics a lot of hypothesis testing is based on the premise of random mating in a given population, so PV92 has been used for a lot of human population genetics studies.
There is another nice thing about PV92. Some people have an Alu sequence inserted in the very middle of the PV92 locus. This Alu insert, an example of an intron (a DNA fragment found in between coding regions of a gene, that doesn't code for any thing and that it's spliced during transcription in functional genes), makes pV92 300 bases longer, which is a difference big enough that it can be visualized in a gel (originally PV92 is about 600 base pairs long).
An Alu sequence is a retrotransposon (a DNA fragment that has been inserted in a location where it doesn't belong via reverse transcription, i.e. mRNA actually writes a sequence into the genome) found throughout primate genomes. There are thousands of copies of Alu sequences found in a single genome.
PCR, the Polymerase Chain Reaction, a method to amplify (make many copies of) specific DNA fragments, is used to generate enough DNA that it can be studied with a variety of thechniques. Using PCR we will amplify our PV92 loci to see if we have the Alu sequence insert in any of our copies of PV92. Each one of us may have 0, 1, or 2 alu inserts in our PV92 copies and we will find out through PCR and gel electrophoresis.
Today we extracted our own DNA. We scraped our cheeks to get some epithelial cells, we neutralized ions (with the InstaGel) that may be used as cofactors by DNases to chop out DNA, and then we bursted our cells open (with the water baths) to extract the DNA from the nuclei.
After lab I added your DNA and mine, plus three positive controls, to a master mix with all the constituents of a PCR: buffer, nucleotides, Taq polymerase, magnesium, and primers (synthesized single stranded oligonucleotide sequences) and put the tubes into the thermocycler. We'll talk about details of how PCR works later.
Tomorrow we'll run a gel electrophoresis to figure out who among us has the Alu insert in his/her PV92 locus, and if it is a single copy or two.
-------------
Tuesday, September 16, 2008
A first glance at molecules within the cell
Today we covered mainly material between pages 1 and 8 in the textbook. We talked about the universality of the hereditary information code (mostly DNA), what are its building blocks, how it replicates within the cell, it is transcribed into RNA, and how it is translated into proteins. In a few words, we went over how a gene is expressed.
We talked at a very basic level, and some of these topics will be revisited in the future and explored further.
On Monday (Sep 22): We'll talk about the secondary structure of RNA, some details of how it is translated into proteins, and what are some of the most common functions of such molecules.
We'll also talk about the implication of studying DNA at the genomic level (again, a very basic, introductory talk).
Reading material: Pages 15-24 of the textbook.
Today's quiz Q&As
1. What is templated polymerization?
The specific mechanism through which DNA replicates, using a strand of DNA as a template to the other
2. What are the two main processes that result in the synthesis of a protein that is encoded in the DNA?
Transcription and translation
3. What is a polypeptide?
A chain of amino acids (All proteins are polypeptides, not all polypeptides are proteins. Not to be confused with polynucleotide, a chain of nucleotides)
4. What is a codon?
A triplet of nucleotides that act as a coding unit for a specific amino acid, or as signals to start and stop the transcription of a gene
5. What is the main function of proteins in a cell?
They are mainly catalysts for a huge veriety of reactions.
(Yes, they are also the building blocks, and agents of cellular organelle repair, but most of them are catalysts of some sort)
3. What is a polypeptide?
A chain of amino acids (All proteins are polypeptides, not all polypeptides are proteins. Not to be confused with polynucleotide, a chain of nucleotides)
4. What is a codon?
A triplet of nucleotides that act as a coding unit for a specific amino acid, or as signals to start and stop the transcription of a gene
5. What is the main function of proteins in a cell?
They are mainly catalysts for a huge veriety of reactions.
(Yes, they are also the building blocks, and agents of cellular organelle repair, but most of them are catalysts of some sort)
Monday, September 15, 2008
The importance of Molecular Biology - topics for review papers
Today we had our first quiz (using the bluebooks), talked a little about the importance of molecular biology, and browsed over a brochure called "Genomics and its impact on science and society", put together by the U.S. Department of Energy Office of Science (see me if you didn't get one). The brochure has an interesting bulleted list on page 7 about applications of molecular biology in different fields.
Also, students teamed up in pairs to propose potential topics for their lieterature review paper in some aspect of molecular biology. We went over some of the poropsoed topics and next week I'll meet with each team in my office (preferably during office hours. Make an appointment so you don't have to wait if I am meeting with another team) to go over your potential topics, your motivation for choosing them, your justification, and to select one of them for your paper.
Q&A's for today's quiz:
1. What are the building blocks of a nucleotide?
Phosphate, sugar, and base
2. What are the processes that result in the synthesis of a protein encoded in the DNA?
Transcription and translation
3. How are new genes generated? (a brief generic explanation. No need to list the specific processes)
My modification of a pre-existing gene (e.g. mutation, duplication, segment shuffling, or horizontal transfer)
4. Mention one way in which the function of a gene can be revealed
By studying its sequence and comparing it to previsouly known ones, by studying the effect of mutations, or by knocking-out genes
5. What is a model organism?
An easy to breed, intensively studied genetically, and therefore well known organism. Conclusions from its study can be extrapolated to other organisms (e.g., the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the mouse Mus musculus, or the thale cress Arabidopsis thaliana)
Also, students teamed up in pairs to propose potential topics for their lieterature review paper in some aspect of molecular biology. We went over some of the poropsoed topics and next week I'll meet with each team in my office (preferably during office hours. Make an appointment so you don't have to wait if I am meeting with another team) to go over your potential topics, your motivation for choosing them, your justification, and to select one of them for your paper.
Q&A's for today's quiz:
1. What are the building blocks of a nucleotide?
Phosphate, sugar, and base
2. What are the processes that result in the synthesis of a protein encoded in the DNA?
Transcription and translation
3. How are new genes generated? (a brief generic explanation. No need to list the specific processes)
My modification of a pre-existing gene (e.g. mutation, duplication, segment shuffling, or horizontal transfer)
4. Mention one way in which the function of a gene can be revealed
By studying its sequence and comparing it to previsouly known ones, by studying the effect of mutations, or by knocking-out genes
5. What is a model organism?
An easy to breed, intensively studied genetically, and therefore well known organism. Conclusions from its study can be extrapolated to other organisms (e.g., the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, the mouse Mus musculus, or the thale cress Arabidopsis thaliana)
Subscribe to:
Posts (Atom)