DNA Technology: BACTERIAL TRANSFORMATION Ms. Gaynor Honors Genetics

What is Bacterial Transformation? Transformation –“Naked” Plasmids (present in environment) are taken up by certain bacteria –Viruses are NOT used in this method! hill.com/sites/ /s tudent_view0/chapter13/ani mation_quiz_1.html

Host E.coli cell is transformed  bacteria takes in plasmid from environment

Bacterial Transformation Step 1 DNA Isolation –Isolation of the “Gene of Interest” (foreign DNA) Step 2 Recombinant DNA –Insertion of foreign DNA into bacterial plasmid using restriction enzymes and DNA ligase Step 3 Transformation –Insertion of recombinant DNA into bacteria by making bacteria competent (weaken) Use CaCl 2 and heat shock techniques

How do you make Bacteria competent? Step 1: Add Calcium Chloride (CaCl 2 ) –CaCl 2 is in a solution (creates Ca +2 and Cl - ions) –DNA in plasmid is negatively charged due to phosphate groups in the backbone –Cell membrane of E. coli also is negatively charged because phospholipids are made of same phosphate groups (PO 4 -3 ) –Ca +2 ions neutralize charges so plasmid can get near (and inside) bacterial cell.

How do you make Bacteria competent? Step 2: Use Heat Shock –Heat Shock is a process that uses warm water (bath) and ice to help get plasmid inside cell Add recombinant plasmid + host cell + CaCl 2 solution to ice then heat then back on ice –Heat = increases kinetic energy of matter Molecules/atoms move faster –Ice = decreases kinetic energy of matter Molecules/atoms mo ve slower mation2.htmlhttp:// mation2.html

DNA Technology: GEL ELECTROHPHORESIS Ms. Gaynor Honors Genetics

DNA Gel Electrophoresis **Each band that you see is a collection of millions of DNA molecules, all of the same length!! Restriction Fragment Analysis detects DNA differences that affect restriction sites DNA fingerprint

Gel electrophoresis Separates DNA restriction fragments of different lengths Uses electrical current to separate DNA based on size DNA has a negative charge. DNA moves towards the POSITIVE electrode. Why? DNA molecules of SMALLER sizes move the furthest through the gel.

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Restriction Fragment Analysis Is useful for comparing two different DNA molecules, such as two alleles for a gene hill.com/sites/ /student _view0/chapter16/animations.html # hill.com/sites/ /student _view0/chapter16/animations.html # Normal  -globin allele Sickle-cell mutant  -globin allele 175 bp 201 bpLarge fragment DdeI 376 bp Large fragment DdeI restriction sites in normal and sickle-cell alleles of  -globin gene. Electrophoresis of restriction fragments from normal and sickle-cell alleles. Normal allele Sickle-cell allele Large fragment 201 bp 175 bp 376 bp (a) (b)

Agarose Gel Electrophoresis 1.Widely used technique for the analysis of DNA (or RNA or proteins) 2.Routinely used (crime scenes, maternity/paternity cases, etc) 3.Separates molecules based on their rate of movement through a gel under the influence of an electrical current 4.We will be using agarose gel (NOT agar)

 To separate a mixture of DNA fragments by size using an electrical charge  The gel is a protein matrix (like a sponge with holes; DNA travels through “holes”) Scanning Electron Micrograph of Agarose Gel (1×1 µm)  Polymerized agarose is porous, allowing for the movement of DNA Purpose of Agarose Gel Electrophoresis

How does gel electrophoresis separate DNA fragments? Gel acts as a strainer to filter DNA by size DNA fragments are naturally negatively charged due to the phosphate backbone (PO 4 -3 ) DNA fragments of differing sizes will move though the gel at differing rates –larger fragments (more bases) = do not travel as far from wells –smaller fragments (less bases) = travel farther from wells

Movement depends on Charge DNA is negatively charged (because of phosphate backbone) DNA will be attracted to positively charged poles and repelled from negatively charged ones

+- Power DNA Small DNA move faster than larger pieces DNA Gel electrophoresis separates DNA according to size Power source supplies the electrical current small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight. Movement Depends on Size

Restriction Enzymes and Plasmid Mapping

Restriction Enzyme Digest  different length pieces are made

Gel electrophoresis markers (called standards or ladders ) are used for size identification of each DNA fragment Each well/column is a “DNA fingerprint”

Casting tray  Gel combs  Power supply  Gel tank   Cover Electrical leads  Gel Electrophoresis Equipment

Making an Agarose Gel And Setting up your Gel Electrophoresis Apparatus

Agarose: Agarose is a linear polymer extracted from seaweed. D-galactose3,6-anhydro L galactose Sweetened agarose gels have been eaten in the Far East since the 17th century. Agarose was 1 st used in biology when Robert Koch used it as a culture medium for Tuberculosis bacteria in 1882 Can be used to separate DNA fragments > 300 bp

An agarose gel is prepared by combining agarose powder and a buffer (ions + H 2 o) solution into a flask. Agarose  Buffer  Flask for boiling 

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

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 B.

Gel casting tray & combs Cast (make) the gel using this tray and comb C.

Seal the edges of the casting tray and put in one comb with 13 teeth. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray. Preparing the Casting Tray C. COMBS CREATE WELLS!!!

Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubble, why? Pouring the gel D.

Make sure that the gel combs are submerged in the melted agarose solution but not touching the bottom. D.

When cooled, agarose polymerizes, forming a flexible gel. It appears cloudy in color when completely cooled (~20 minutes). Carefully remove comb (be very, very careful…don’t remove at an angle!). E.

Place the gel in the electrophoresis chamber.

buffer  Add enough buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer. Buffer allows electrical current to FLOW through chamber!  Cathode (negative end) BLACK WIRE! Anode  (positive end) RED WIRE!  wells  DNA 

REVIEW…Loading and Running the gel Molten agarose is poured into a casting tray and a comb is placed inside the casting tray. After the agarose solidifies, the comb is removed leaving wells where the DNA will be loaded. DNA samples are mixed with tracking dye which contains glycerol (to weigh down the DNA into the well ) and acts as a mobile dye so that you can visualize migration –this is why the DNA “falls” into the wells and you can SEE it move through the gel!!! A buffer containing ions (to conduct an electric current) is placed in the chamber around the gel after

Loading Dye:  FUNCTIONS:  Bromophenol Blue (for color)  Glycerol (for weight) Sample Preparation Samples of DNA need to be mixed with tracking dye. Allows DNA samples to be seen in the gel Increases the density of samples, causing them to sink into the gel wells.

Loading the Gel Carefully place the micropipette tip over a well and gently expel the sample. The sample should sink into the well NOT float in the buffer. Be careful not to puncture the gel with the pipette tip.

Place the cover on the electrophoresis chamber and connect the electrical leads. 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 (-) End Anode (+) End 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 (-)  Migration

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. YOU ARE USING A QUICK DNA STAIN!!! 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.

Staining the Gel Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for minutes. To remove excess stain, allow the gel to destain in water. Replace water several times for efficient destain.

Staining the Gel Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for minutes. To remove excess stain, allow the gel to destain in water. Replace water several times for efficient destain.

Methylene blue requires an ultraviolet light source to visualize

Visualizing the DNA  100  200  300  1,650  1,000  500  850  650  400  5,000 bp  2,000 DNA ladder  DNA ladder/Size standard  DNA: wells  Samples # 1, 4, 6 & 7 were positive for DNA samples taken from the crime and compared to suspect

Visualizing the DNA (Actual Image)  250  1,500  1,000  500  750  2,000 bp DNA ladder  DNA wells  March 12, 2006 Samples # 1, 6, 7, 10 & 12 were positive for our suspect and crime scene samples

Movement of DNA fragments in agarose gels There is a linear relationship between the migration rate of a given DNA fragment and the logarithm of its size (in basepairs). Larger molecules move more slowly through the gel because of more friction

Semilog paper

Distance migrated (mm) Fragment Length (bp) GRAPH THE LADDER/STANDARD…then make a best fit line or curve!

Distance migrated (mm) Fragment Length (bp) x bp