Molecular Biology

(MLMB-201)

Lecturer:Dr. Mohamed Salah El-Din

Department of Medical Laboratory TechnologyFaculty of Allied Medical Science

Intended Learning Outcomes (ILO’s):

Molecular biology course 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 ElectrophoresisGel Electrophoresis

The questionsThe questions

Why? To separate pieces of DNA by size.Why? To separate pieces of DNA by size.

How? Use electrical charge to pull the How? Use electrical charge to pull the negatively charged DNA through a gel that has negatively charged DNA through a gel that has small pores in it.small pores in it.

When? When doing DNA fingerprinting, When? When doing DNA fingerprinting, analysis of plasmids, determining sizes of analysis of plasmids, determining sizes of fragments, mapping plasmidsfragments, mapping plasmids

• DNA is negatively charged.

+-

Power

DNA

• When placed in an electrical field, DNA will migrate toward the positive pole (anode).

H

O2

• An agarose gel is used to slow the movement of DNA and separate by size.

Scanning Electron Micrograph

of Agarose Gel (1×1 µm)

• Polymerized agarose is porous,

allowing for the movement of DNA

+-

Power

DNA

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

smalllarge

Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

BufferBuffer

Buffer is to keep the pH constant.Buffer is to keep the pH constant.

If the pH changed the HIf the pH changed the H++ charges would be charges would be pulled to the negative cathode and would set pulled to the negative cathode and would set up a pH gradient in the box.up a pH gradient in the box.

Agarose

Agarose is a linear polymer extracted from seaweed.

D-galactose 3,6-anhydroL-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 vs AgarAgarose vs Agar

Agar: from seaweed is food for bacteria to Agar: from seaweed is food for bacteria to grow upongrow upon

Agarose: sugar gel for doing electrophoresisAgarose: sugar gel for doing electrophoresis

Making an Agarose Gel:

An agarose gel is prepared by combining agarose powder and a buffer solution.

Agarose

Buffer

Flask for boiling

Casting tray

Gel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

Gel casting tray & combs

Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

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.

Melting the Agarose

How thick?How thick?

Agarose is measured in %, which is Agarose is measured in %, which is grams per 100ml of solution.grams per 100ml of solution.

A 7% gel vs a 3 % gel;A 7% gel vs a 3 % gel; Thicker so it is easier to hold without Thicker so it is easier to hold without

breakingbreaking Slower for molecules to go through and Slower for molecules to go through and

hard for big molecules to make it.hard for big molecules to make it.

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

Pouring the gel

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.

Place the gel in the electrophoresis chamber.

buffer

Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

Cathode(negative)

Anode(positive)

wells

DNA

6X Loading Buffer: Bromophenol Blue (for color) Glycerol (for weight)

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.

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.

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.

Running the Gel

wells Bromophenol Blue

Cathode(-)

Anode(+)

Gel

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.

DNA(-)

100 200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000

DNA Ladder Standard

Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

-

+

DNAmigration

bromophenol blue

Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule

Staining the Gel

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

• 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.

Safer alternatives to Ethidium Bromide

Methylene Blue

BioRAD - Bio-Safe DNA Stain

Ward’s - QUIKView DNA Stain

Carolina BLU Stain

…others

advantagesInexpensiveLess toxicNo UV light requiredNo hazardous waste disposal

disadvantagesLess sensitiveMore DNA needed on gelLonger 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.

Ethidium Bromide requires an ultraviolet light source to visualize

Visualizing the DNA (ethidium bromide)

100 200 300

1,650 1,000

500

850 650

400

5,000 bp 2,000

DNA ladder

DNA ladder

PCR Product

1 2 3 4 5 6 7 8

wells

+ - - + - + + -

Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA

Primer dimers

Protein Electrophoresis and SDS-PageProtein Electrophoresis and SDS-Page

A Quick Intro to the SDS-PAGEA Quick Intro to the SDS-PAGE

Proteins are separated using polyacrylamide Proteins are separated using polyacrylamide gels rather than agarose gels, because the gels rather than agarose gels, because the polyacrylamide matrix polyacrylamide matrix

A)A) have have pore sizespore sizes similar to the sizes of proteins similar to the sizes of proteins (DNA is usually larger in size)(DNA is usually larger in size)

B)B) are much are much tightertighter than agarose gels, thus are than agarose gels, thus are able to resolve the smaller protein moleculesable to resolve the smaller protein molecules

A Quick Intro into the SDS-PageA Quick Intro into the SDS-Page Stands for Stands for sodium dodecyl sulfate sodium dodecyl sulfate

polyacrylamide gel electrophoresispolyacrylamide gel electrophoresis Separates proteins according to Separates proteins according to molecular molecular

weightweight Prior to electrophoresis, proteins are treated with Prior to electrophoresis, proteins are treated with

a detergent solution, the sodium dodecyl sulfate, a detergent solution, the sodium dodecyl sulfate, and heatedand heated

SDS and HeatSDS and Heat

The SDS and heat denatures the proteins’ secondary, The SDS and heat denatures the proteins’ secondary, tertiary, and quaternarytertiary, and quaternary

structures →sometimes reducing agent is added to break structures →sometimes reducing agent is added to break disulfide bonds (Why?)disulfide bonds (Why?)

http://www.molecularstation.com/images/protein-gel-electrophoresis.jpg

More About SDSMore About SDS

In addition to linearlizing the protein structure, In addition to linearlizing the protein structure, SDS also gives the protein an overall negative SDS also gives the protein an overall negative charge with a strength that is relative to the charge with a strength that is relative to the length of the protein.length of the protein.

http://media.wiley.com/CurrentProtocols/ET/et0703/et0703-fig-0001-1-full.gif

The PAGEThe PAGE

Uses two phases of polyacrylamide:Uses two phases of polyacrylamide:

1)1) An upper An upper stacking gelstacking gel that is usually 4% that is usually 4% acrylamide→ allows the proteins to migrate acrylamide→ allows the proteins to migrate rapidly and concentrates them into uniform rapidly and concentrates them into uniform bands before hitting the denser gelbands before hitting the denser gel

2)2) A lower A lower separating gelseparating gel that is of a higher that is of a higher percentage of acrylamide (usually percentage of acrylamide (usually 15%)→separates according to molecular 15%)→separates according to molecular weightweight

The PAGEThe PAGE

http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg

What is in the gel?What is in the gel?

SDSSDS Tris-HCl bufferTris-HCl buffer DTT (Reducing agent)DTT (Reducing agent) AcrylamideAcrylamide BisacrylamideBisacrylamide Ammonium persulfate and TEMED are Ammonium persulfate and TEMED are

added when the gel is ready to polymerizeadded when the gel is ready to polymerize

What is in the buffer?What is in the buffer?

SDS-PAGE running bufferSDS-PAGE running buffer Made with Tris and glycine Made with Tris and glycine The chlorine ions (gel) migrate much more The chlorine ions (gel) migrate much more

rapidly then the glycine ions (buffer) with rapidly then the glycine ions (buffer) with the proteins having a migration speed in the proteins having a migration speed in between the two ions→ proteins become between the two ions→ proteins become trapped in a narrow band between the two trapped in a narrow band between the two ion fronts when electrophoresis begins..ion fronts when electrophoresis begins..

The ApparatusThe Apparatus

http://www.gbiosciences.com/EducationalUploads/EducationalProductsImages/mediumimages/Protein%20Electrophoresis.jpg

http://www.nda-analytics.com/images/pipetting.jpg

The SetupThe Setup

http://en.wikipedia.org/wiki/SDS-PAGE

The SeparationThe Separation As soon as the electric current is applied, the SDS coated proteins begin As soon as the electric current is applied, the SDS coated proteins begin

their journey towards the positive electrode.their journey towards the positive electrode. Smaller proteins migrate more quickly than larger proteins through the Smaller proteins migrate more quickly than larger proteins through the

polyacrylamide pores polyacrylamide pores

http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg

Visualizing your Protein during Visualizing your Protein during ElectrophoresisElectrophoresis

To show the progress of your electrophoresis, a To show the progress of your electrophoresis, a blue tracking dyeblue tracking dye is mixed in with your protein is mixed in with your protein samples.samples.

The blue dye is negatively chargedThe blue dye is negatively charged

The blue dye is smaller than the proteins The blue dye is smaller than the proteins expected in your sample, so they slightly move expected in your sample, so they slightly move ahead of the proteins in the gel. ahead of the proteins in the gel. Why is this Why is this important?important?

Visualizing your Protein after Visualizing your Protein after ElectrophoresisElectrophoresis

The gel is stained with The gel is stained with Coomassie Brilliant BlueCoomassie Brilliant Blue R-250R-250

This stain binds specifically to proteins and no This stain binds specifically to proteins and no other molecule, such as DNA, carbohydrates, or other molecule, such as DNA, carbohydrates, or lipids.lipids.

After staining, distinct blue bands appear on the After staining, distinct blue bands appear on the gel based on how much protein is in that bandgel based on how much protein is in that band

The larger the amount of protein, the more The larger the amount of protein, the more intense the stainingintense the staining

http://parts.mit.edu/igem07/index.php/Berkeley_LBL/Results/ProteinGel

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.