Molecular Biology (MLMB-201) Department of Medical Laboratory Technology Faculty of Allied Medical Science Lecturer: Dr. Mohamed Salah El-Din
Intended Learning Outcomes (ILO’s): • Molecular biologycourse provides an overview of the molecular basis to cell structure and function. • This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and how these interact among themselves and with proteins. Molecular biology techniques are essential for modern biological and medical research. This course will give you an introduction to DNA and RNA standard techniques. • Student will have basic knowledge of: • Cell organization. • DNA structure and function. • DNA Extraction. • RNA structure and function. • RNA Extraction. • Gene expression and protein biosynthesis. • Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein. • Polymerase Chain Reaction (PCR) – Theory, Types, Application. • Gene library and screening • DNA sequencing
Gel Electrophoresis The questions • Why? To separate pieces of DNA by size. • How? Use electrical charge to pull the negatively charged DNA through a gel that has small pores in it. • When? When doing DNA fingerprinting, analysis of plasmids, determining sizes of fragments, mapping plasmids
• When placed in an electrical field, DNA will migrate toward the positive pole (anode). H á O2 á DNAà - + Power • Polymerized agarose is porous, allowing for the movement of DNA Scanning Electron Micrograph of Agarose Gel (1×1 µm) à •DNA is negatively charged. • An agarose gel is used to slow the movement of DNA and separate by size.
DNA - + Power How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
Buffer Buffer is to keep the pH constant. If the pH changed the H+ charges would be pulled to the negative cathode and would set up a pH gradient in the box.
Agarose D-galactose 3,6-anhydro L-galactose • Sweetened agarose gels have been eaten in the Far East since the 17th century. • Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882 *Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteria Agarose is a linear polymer extracted from seaweed.
Agarose vs Agar • Agar: from seaweed is food for bacteria to grow upon • Agarose: sugar gel for doing electrophoresis
Making an Agarose Gel: An agarose gel is prepared by combining agarose powder and a buffer solution. BufferØ Flask for boilingÙ AgaroseÚ
Electrophoresis Equipment Power supplyà åCover Gel tankà Electrical leads â Casting trayä Gel combsä
Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.
Melting the Agarose Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.
How thick? • Agarose is measured in %, which is grams per 100ml of solution. • A 7% gel vs a 3 % gel; • Thicker so it is easier to hold without breaking • Slower for molecules to go through and hard for big molecules to make it.
Pouring the gel Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.
Each of the gel combs should be submerged in the melted agarose solution.
When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.
DNAè bufferè á á á á wells Anodeä (positive) ãCathode (negative) Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.
Sample Preparation Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, as it has glycerol added to increase the density to greater than water. This causes the dye and DNA sample to sink into the gel wells. Remember the wells are like little cups, open at the top and walls on each side. 6X Loading Buffer: à · Bromophenol Blue (for color) · Glycerol (for weight)
Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.
Running the Gel Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.
Cathode (-) ß wells ß Bromophenol Blue DNA (-) â Gel Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.
ß 12,000 bp ß 5,000 DNA migration ß 2,000 ß 1,650 ß 1,000 ß 850 ß 650 ß 500 ß 400 ß 300 ß 200 ß 100 DNA Ladder Standard - Note:bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule bromophenol blueà + Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.
Staining the Gel •Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. • Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run. ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.
Safer alternatives to Ethidium Bromide • · Methylene Blue • · BioRAD - Bio-Safe DNA Stain • Ward’s - QUIKView DNA Stain • Carolina BLU Stain • …others advantages Inexpensive Less toxic No UV light required No hazardous waste disposal disadvantages Less sensitive More DNA needed on gel Longer staining/destaining time
Staining the Gel • Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 25-30 minutes. • To remove excess stain, allow the gel to destain in water. • Replace water several times for efficient destain.
DNA ladder â DNA ladder â 1 2 3 4 5 6 7 8 wellsà • 5,000 bp ß 2,000 ß 1,650 ß 1,000 ß 850 ß 650 ß 500 ß 400 PCR Product ß 300 ß 200 ß 100 + - - + - + + - Visualizing the DNA (ethidium bromide) Primer dimersà Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA
Protein Electrophoresis and SDS-Page A Quick Intro to the SDS-PAGE Proteins are separated using polyacrylamide gels rather than agarose gels, because the polyacrylamide matrix • have pore sizes similar to the sizes of proteins (DNA is usually larger in size) • are much tighter than agarose gels, thus are able to resolve the smaller protein molecules
A Quick Intro into the SDS-Page • Stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis • Separates proteins according to molecular weight • Prior to electrophoresis, proteins are treated with a detergent solution, the sodium dodecyl sulfate, and heated
SDS and Heat • The SDS and heat denatures the proteins’ secondary, tertiary, and quaternary structures →sometimes reducing agent is added to break disulfide bonds (Why?) http://www.molecularstation.com/images/protein-gel-electrophoresis.jpg
More About SDS • In addition to linearlizing the protein structure, SDS also gives the protein an overall negative charge with a strength that is relative to the length of the protein. http://media.wiley.com/CurrentProtocols/ET/et0703/et0703-fig-0001-1-full.gif
The PAGE Uses two phases of polyacrylamide: • An upper stacking gel that is usually 4% acrylamide→ allows the proteins to migrate rapidly and concentrates them into uniform bands before hitting the denser gel • A lower separating gel that is of a higher percentage of acrylamide (usually 15%)→separates according to molecular weight
The PAGE http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg
What is in the gel? • SDS • Tris-HCl buffer • DTT (Reducing agent) • Acrylamide • Bisacrylamide • Ammonium persulfate and TEMED are added when the gel is ready to polymerize
What is in the buffer? SDS-PAGE running buffer • Made with Tris and glycine • The chlorine ions (gel) migrate much more rapidly then the glycine ions (buffer) with the proteins having a migration speed in between the two ions→ proteins become trapped in a narrow band between the two ion fronts when electrophoresis begins..
The Apparatus http://www.gbiosciences.com/Educational Uploads/EducationalProductsImages/mediumimages/Protein%20Electrophoresis.jpg http://www.nda-analytics.com/images/pipetting.jpg
The Setup http://en.wikipedia.org/wiki/SDS-PAGE
The Separation • As soon as the electric current is applied, the SDS coated proteins begin their journey towards the positive electrode. • Smaller proteins migrate more quickly than larger proteins through the polyacrylamide pores http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg
Visualizing your Protein during Electrophoresis • To show the progress of your electrophoresis, a blue tracking dye is mixed in with your protein samples. • The blue dye is negatively charged • The blue dye is smaller than the proteins expected in your sample, so they slightly move ahead of the proteins in the gel. Why is this important?
Visualizing your Protein after Electrophoresis • The gel is stained with Coomassie Brilliant Blue R-250 • This stain binds specifically to proteins and no other molecule, such as DNA, carbohydrates, or lipids. • After staining, distinct blue bands appear on the gel based on how much protein is in that band • The larger the amount of protein, the more intense the staining
Assignment: As a part of the semester activity, a group of students is selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.