Chapter 21: Techniques of Molecular Biology
I. Introduction
A. Introduction: Developing Molecular Techniques To Study Cellular Function
1. Gene expression is necessary for the cell to function properly
a. DNA à RNA à Protein
b. The protein is what carries out the function of the gene with the information encoding each protein housed within the DNA
c. Each step of this process is regulated
2. Types of proteins produced:
a. Enzymes to carry out important biological reactions
b. Structural proteins to give the cell shape
c. Membrane bound proteins to receive signals and to anchor the cell
d. Signaling proteins which are secreted and carry signals from one cell to another
f. Other proteins not covered in the other four categories
3. In order to study each step in the process, Molecular Biologists needed to develop techniques to study each molecule in this complicated process
B. Introduction: Practical Reasons For Developing Molecular Techniques
1. Techniques in Molecular Biology have been developed for four reasons
a. Study basic gene function/expression
b. Determine root causes of human genetic diseases and the symptoms that result
c. Disease diagnotics
d. Forensics
2. Molecular biologists can easily follow two types of classes of genetic diseases
a. Chromosomal disorders
b. Single gene disorders
3. Chromosomal disorders:
a. An individual has too many copies of a single chromosome
b. Ex. Downs Syndrome
c. Tools needed are those to visualize chromosomes in situ or in vitro
4. Single gene disorders
a. Disorders resulting from a mutation in a single gene
b. Need techniques to study the DNA encoding the implicated gene
c. Need techniques to study the corresponding molecules produced when the implicated gene is expressed
C. Introduction: Practical Reasons For Developing Molecular Techniques
1. Besides these experimental tools being used for the basic study of gene function and the root cause of genetic disease, many have been either used, or modified such that they can be used to diagnose disease
a. Can be used to confirm or determine whether one has disease causing alleles
b. Can be used for preventative purposes
c. These techniques are used “genotype” people
2. Many of the same techniques used in disease diagnosis are also used in forensics due to the fact that one also wants to “genotype” suspects and criminals to find out who committed the crime
II. Visualizing Molecules
A. Visualizing Molecules: Introduction
1. To study genes/gene expression/genetic disease on a molecular, techniques were developed to visualize the following molecules
a. DNA
b. RNA
c. Protein
2. The methods by which to visualize DNA and RNA are quite similar because they have similar chemistry
a. Nucleic acids
b. Negatively charged
c. Same directional polarity
d. Only difference is that one is double stranded and the other is single stranded
3. Visualization methods for proteins can be quite different due to the fact that they have a different chemistry than DNA and RNA
B. Visualizing Molecules: DNA and RNA
1. Agarose gel electrophoresis is used to study nucleic acids
a. DNA
b. RNA
2. The agarose is an inert substance that is used to create a porous gel matrix, which allows us to separate DNA molecules according to size
3. To produce an agarose gel, the agarose powder is mixed into solution with either 1X TAE or 1X TBE buffer
a. Buffers allow for the dissolving of agarose when heat is applied
b. Buffers contain salts which are necessary for running your nucleic acid into the gel
4. Once the agarose is melted into solution, the mixture is poured into a tray and allowed to solidify creating a matrix
a. DNA molecules are moderately flexible and occupy effective volume
b. The gel matrix acts as a sieve through which DNA molecules pass
c. Pore size is dependent on the % of agarose in the gel matrix
d. Large DNA molecules have more volume and thus have more difficulty passing though the gel than smaller DNA molecules and so they run smaller
5. The gel is placed into a gel box, and the box is also filled with the appropriate salt buffer
a. If gel contains 1X TAE, then 1X TAE is used
b. If gel contains 1X TBE, then 1X TBE is used
6. In order to run your DNA samples through the gel, they are placed in wells at the top of the gel
7. Electrical current is applied-with the buffer salts being able to carry it through the chamber/gel
a. Negative electrode (black) is placed near the top of the gel
b. Positive electrode (red) is placed near the bottom of the gel
c. Since the DNA is negatively charged it will run toward the positive electrode when current is applied
8. Smaller DNA molecules will run faster than large ones
9. In order to visualize the DNA, the gel is stained with ethidium bromide
a. Interacalating agent
b. DNA fluoresces when UV light is shine on the gel
c. DNA will be visualized as bands
10. When running an agarose gel, a size standard is also used for comparison to determine the size of the DNA bands in your samples
11. RNA is also visualized also using agarose gels
a. RNA has secondary structure
b. Gel will contain agarose, buffer and formaldehyde
c. Formaldehyde is used to denature the secondary structure in the RNA to ensure it runs true to size
C. Visualizing Molecules: Proteins
1. In order to visualize proteins, we also use gel electrophoresis
2. To visualize proteins, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used instead of agarose gel electrophoresis
a. Acrylamide will form the gel matrix
b. SDS acts as a denaturant to relieve structure within the protein so that it runs true to size on the gel
c. SDS also coats the linearized proteins (which have a relatively uncharged backbone) giving them a net negative charge
3. The SDS-Polyacrylamide gel, actually consists of 2 gels which consists of different amounts of acrylamide
a. Stacking gel (which stacks all the proteins so the enter the separating gel together
b. Separating gel, which allows for separation of proteins according to size
4. A running buffer of Tris/Glycine and SDS is used
5. Since the protein now has a net negative charge, electrical current can be used to run the proteins through the gel
a. Negative electrode is placed at the top of the gel
b. Positive electrode is placed at the bottom of the gel
6. Proteins run on the SDS-Polyacrylamide gel according to size
a. Proteins with greater molecular weight (larger proteins) run slower
b. Proteins with lesser molecular weight (smaller proteins) run faster
7. To visualize the proteins, coomassie blue dye is used, which binds the proteins and causes them to appear purple
8. Proteins size is measured as a function of molecular weight (Daltons)
10. A size standard (ladder) is run as a guide to determine the molecular weights of proteins in your sample lanes
D. Visualizing Molecules: Proteins by Coomassie Blue Staining
1. If you run purified protein on an SDS-Polyacrylamide gel, then you should see only one band upon coomassie blue staining
a. Band represents the only type of protein present in the sample
b. Many copies of that single protein in the visible band
c. Band should run true to the protein’s size
2. If you run a cellular extract, then you will see all the different proteins present in the cell type
a. Each type of protein is represented by a band on the gel
b. Each protein band should run true to size
E. Visualizing Molecules: Finding A Single Protein From A Cellular Extract
1. Advantages of coomassie blue staining of an SDS-Polyacrylamide
a. Easy
b. Can see all the proteins present in the cellular extract
2. If you want to study one specific protein in a cellular extract, coomassie blue staining of the gel is not the best way to visualize
a. Your protein will be one band amongst many
b. An extract may contain several proteins of similar molecular weight
3. The western blot can be used as a technique to study a single protein in an extract
a. Whether the protein is present in an extract, and if so, the gene is expressed
b. Western blot, if done right, can tell you protein levels-which will give an idea to the level the gene is expressed
F. Visualizing Molecules: Creating Protein Specific Antibodies For Western Blots
1. The western blot technique requires antibodies
a. Produced by B-cells
b. Antibodies are a specific type of protein
c. Are meant to recognize foreign proteins in vivo
2. Production of antibodies:
a. For the protein you are interested in studying, first obtain purified protein
b. Inject the purified protein into a rabbit
c. Rabbit’s immune system will recognize that the protein is foreign and will make antibodies against the protein
d. Take blood samples from the rabbit and then purify antibodies from the blood samples
e. Antibodies produced are considered polyclonal antibodies because each protein that is injected a mixture of antibodies will made that recognize different epitopes within the protein
G. Visualizing Molecules: Actual Western Blotting Procedures
1. First, protein must be extracted from the cell line of choice to make an extract
2. Next, a sample of extract is then mixed with SDS-sample buffer and boiled
a. The sample buffer contains dye
b. The sample buffer contains SDS, which is a detergent
c. The SDS acts to denature the protein
d. The SDS also coats the protein, giving it a net negative charge
f. The boiling step also aids in the denaturation of the protein
3. Once the proteins are denatured, the sample above is run on an SDS-polyacrylamide gel
4. Once the gel is run, the proteins are transfer horizontally to a nitrocellulose membrane
a. In the transfer apparatus, the gel is placed vertically to allow for a horizontal transfer
b. Proteins are transferred by using electrical current
c. The transfer is done horizontally towards the anode, as the proteins are coated with the negatively charged SDS
5. Supplemental Figure: Visualizing Molecules: Actual Western
6. In order to visualize your proteins on the blot, the blot must be probed with your antibodies
7. In order to get an efficient probing of the blot, two antibodies are used
a. The primary antibody binds the protein of interest
b. The secondary antibody binds the constant region of the primary antibody
c. The constant region of an antibody is held in common for all antibodies produced by organisms of the same species
d. The secondary antibody has either Alkaline phosphatase or Horseradish peroxidase conjugated to it
8. Once the antibodies are bound, the protein of interest can be visualized by two methods
a. Using a chemiluminescent substrate or a colorimetric substrate
b. The conjugated enzyme will oxidize the substrate and if it is the chemiluminescent substrate, light will be emitted
c. The conjugated enzyme will oxidize the substrate and if it is the colorimetric substrate, then a purple product is left behind
9. In either case, the protein of interest will appear as a band on the blot
10. Besides telling you whether a protein is present or absent in a sample, a western blot can tell you whether the protein’s size or levels has been altered
11. If the protein is somehow truncated, then one would find the protein lower on the western blot (because it ran faster on the gel)
12. If the protein’s levels have been altered, the intensity of the band may be altered
a. If levels of protein are increased then the band appears darker
b. If the levels of protein are decreased, then the band appears lighter
H. Visualizing Molecules: Finding A Specific RNA From An Extract
1. In addition to following proteins, when studying gene expression RNA can be followed
a. mRNA serve as substrates for translation and their levels are oftentimes proportional to the amount of protein produced
b. For some genes, the RNA is the terminal product
2. The northern blot is used to study a single RNA amongst all present in a cellular extract is the northern blot
a. Method is easy and produces results that are reproducible
b. Most common method used to study cellular mRNA levels
c. Inexpensive
d. This technique can be used to determine whether a specific gene is expressed, and to what levels it may be expressed
3. Note: Again for protein coding genes, this technique is not the most direct technique to study gene expression
J. Visualizing Molecules: Northern Blot Analysis-Procedural Overview
1. First RNA isolations must be performed from the cell line/strain of choice
a. Create a sample of cellular RNA (all types)
b. Only ~2% of RNA is mRNA in yeast, ~10% in other species
2. The total RNA is run on a formaldehyde-agarose gel (1.25%)
a. Staining with ethidium bromide will show the ribosomal RNA as bands on the gel
b. The mRNA, since the levels are so low, will not be seen by staining
3. The RNA is transferred to a nylon membrane by capillary action
K. Visualizing Molecules: Transferring the RNA To The Nylon Membrane (agarose gel)