DNA Fingerprinting Information - Angelfire



DNA Fingerprinting Information

DNA fingerprinting (DNA profiling), is a method used in various medical and forensic procedures, as well as in paternity determinations. DNA fingerprinting is used to examine similarities and differences in organisms at the genetic-molecular level. This lesson will simulate the technique most commonly used in human forensics - that is, taking DNA from any body cell, digesting the DNA with restriction enzymes, and analyzing the resulting DNA fragments using agarose gel electrophoresis. The lab results of this activity provide DNA patterns in a gel, which reveal the differences and similarities in an individual’s genetic make-up. This activity also provides a starting point for rich and diverse discussions on the societal, ethical and legal implications of DNA analysis. Many of the revolutionary changes that have occurred in biology over the past fifteen years can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can cut, mend, wind, unwind, transcribe, repress, and replicate DNA. Restriction enzymes are the ‘chemical scissors’ of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone’s DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically ‘chopped up’ into a very different collection of fragments. The DNA fragments are separated by electricity – agarose gel electrophoresis – and tagged or stained in some fashion so that they can be visualized and studied. The resulting pattern of restriction fragments resembles the pricing bar code used on supermarket products – small bars lined up in a column, the large fragments closest to the beginning of the gel, the smallest at the end of the gel.

Methods of DNA identification have been applied to many branches of science and technology, including medicine (prenatal tests, genetic screening), conservation biology (guiding captive breeding programs for endangered species), and forensic science. In the latter discipline, analysis of the pattern of DNA fragments in a restriction digest, loosely called a DNA fingerprint, enables us to discriminate between suspects accused of a crime or potential fathers in a paternity suit.

In DNA fingerprinting, radioactive pieces of DNA called probes are added to the separated DNA fragments. The probes are designed to bind to specific sequences on the DNA, thus marking some of the fragments. X-ray film placed on top of the gel will become exposed by the radioactivity, and black bands will become visible on the film. The variation in DNA from one person to the next is so great that the probability of two people sharing the same DNA fingerprint is essentially zero. And unlike conventional fingerprints, which are often difficult to gather at a crime scene, a DNA fingerprint can be made from a very small sample of blood, skin, or even a single hair.

EXPERIMENT ONE

Background Info

You will be using restriction enzymes to digest five hypothetical samples of human DNA. One sample of DNA is collected from a ‘crime scene’ and four samples are obtained from possible ‘suspects.’ The resulting restriction fragments are separated by agarose gel electrophoresis and visualized using Bio-Safe DNA staining solution. Based on the restriction fragment patterns, you will analyze their results, then match the crime scene sample with that of the hypothetical suspect. (This experiment does not use human DNA). This experiment can also be used to demonstrate the basic procedures of agarose gel electrophoresis; including gel casting, sample application, separation and DNA staining.

Concept

Complex mixtures of molecules can be separated by size, shape, and electric charge using gel electrophoresis.

Objectives

Upon completion of this laboratory exercise, students will be able to:

Use a micropipettor to correctly measure small volumes of liquid molecules using microliter units;

Cast an agarose gel of correct agarose percentage;

Load an agarose gel with specific amounts of material in the wells;

Analyze dye samples by gel electrophoresis and interpret the results;

Identify and manipulate the variables involved in separating biological dye samples;

Explain the theory behind molecular separation of molecules by electrophoresis.

TEACHER COPY

Background on Lab

This lab activity will likely take many class periods to complete if the students prepare and practice loading agarose gel wells the first day.

Kit Materials

2 double gel boxes – photodyne, casting trays, combs

1 power supply

8 micropipettors: 2 - 20 ml

4 containers of yellow tips

1 set of dyes (5 mls of each) in 0.1 volume glycerol

16 plastic rules with graduations in millimeters

50X TAE buffer (80 mls.) Dilute to 1X TAE for gel preparation and for electrophoresis buffer.

1 g of agarose

1.5 ml of microtubes (70)

Permanent marking pens for labeling tubes

Gloves

Thermometer

Water bath at 60 degree Centigrade for holding agarose temperature

Practice loading dyes

First Day

Students will pour practice loading gels in a petri dish bottom and practice loading with any or all of the 3 practice loading dyes.

Second Day

Students will cast the gel tray, set up the gel box, add buffer, load the wells, run the electrophoresis, and read and analyze the electrophoresis results.

Dyes

1. Blue food coloring is – charged. It will move toward the + pole

2. Yellow food color is – charged and will move toward the _ pole.

3. Methylene Blue is + charged and will move toward the – pole.

4. Mystery mixture “A” is composed of safranin and yellow food color. Safranin will migrate towards the – pole and Yellow food color will migrate towards the + pole.

5. Mystery mixture “B” is composed of blue food color and methylene blue. Blue food color will migrate towards the + pole and methylene blue will move toward the – pole.

Samples to be run:

1. Blue food coloring

2. Yellow food coloring

3. Methylene Blue

4. Mixture Mystery A

5. Mixture Mystery B

Loading the gel:

Your teacher will assign six wells for you to load your dye samples.

Load 10 µl of dye “1” into the first well.

Change tip.

Load 10 µl of dye “2” into the 2nd well.

Change tip.

Load 10 µl of dye “3” into the 3rd well, etc.

When all groups have finished loading, connect the leads to the electrophoresis box (Black to black and red to red). Attach the other set of leads to the power supple (red to red, black to black)

Run the electrophoresis system of 10 minutes at 100 volts.

Turn off the power. Note which end of the gel was connected to the Positive (_, red, anode) pole. Remove gel tray and lightly blot dry with a paper towel.

Carefully remove the gel and place it on a piece of paper toweling on the lab table.

Using a marker pen, mark lines for each well and for each dye spot on the gel in their exact locations and indicate colors on your paper. Note the positive electrode end.

STUDENT COPY

Preparing and casting agarose gels

Objectives:

Students will be able to prepare an agarose gel for gel electrophoresis in the correct concentration.

Materials:

centigram balance

Weighing boats of paper

Agarose powder

1X TAE buffer

gel casting tray and comb

4 practice combs

3 – 4 petri plates

Procedure:

First Day:

Making the Agarose solution:

Measure 30 mls of 1X TAE buffer – put into a 250 ml pyrex bottle or Erlenmeyer flask.

Weigh out 0.30 grams of agarose powder. Add to the bottle of TAE buffer and swirl to mix the agarose and buffer.

Place a lid or cork on the top and store at room temperature until day 2.

Making the Practice gel:

Pour liquid agar in a petri dish until it is half full. (Caution! Liquid agar is very hot and can burn. Wear gloves.)

Hold a well comb in the agar until the agar solidifies, or fix the comb so it will stay in place until the agar turns solid. The comb tips should not reach the bottom of the dish!

After the agar has turned solid, pour water over the surface of the gel.

Carefully remove the comb by pulling it straight up while the gel tray is in place.

Practice Loading:

Connect a yellow tip to a p20 micropipettor.

Set the dial on the pipettor at 10 µl

Press plunger to first stop, insert tip into dye sample while slowly releasing the plunger.

Insert tip into water just above the well.

Press plunger slowly to first stop to release dye into well.

Press plunger to final stop as you pull pipettor out of the water.

Repeat at least 10 times.

Second Day

Dissolving the agarose:

Heat to boiling in microwave for 2 minutes (or heat on a hot plate). (CAUTION! Agarose will boil over quite easily!) An Erlenmeyer flask or a bottle with a loosened cap works well. Use the hot gloves or a potholder to protect yourself!

Check bottle – swirl again to see if agarose is dissolved. If not, heat for another 30 seconds and check again. Swirl occasionally.

After the agarose is clear and ALL particles have dissolved, you can keep the agarose liquid at approximately 60 degrees Centigrade in a hot water bath.

Pouring the gel:

Set the dams at the end of the gel casting tray in the upper position and tighten the screws. Place the casting tray on a level spot on the lab table.

Pour 30 ml of melted agarose solution into the casting tray and then insert the well comb in the middle position on the gel bed. Avoid getting air bubbles in the gel.

Let gel cool until it has set, (approximately 10 minutes). The agarose will turn from clear to whitish. It will be firm and look like Jell-O. You are now ready to load the gels, or store them in a plastic baggie or Tupperware container with some 1X TAE buffer until they are needed.

Preparation of the Electrophoresis Apparatus:

Place the gel into the gel box and add enough 1X TAE buffer to just cover the gel to a depth of about 1 – 2 mm. This should take about 800 mls for the large electrophoresis boxes.

EXPERIMENT TWO

TEACHER COPY

Materials: (per team)

Gel electrophoresis box

Agarose, liquid held at 65* C (» 30 ml/gel)

Power supply

Restriction enzymes on ice

Casting tray and comb

DNA samples, on ice

P-20 micropipet and tips

Electrophoresis buffer, » 1X TAE

Racks for 1.5 ml reaction tubes

Restriction buffer [2X]

1.5 ml reaction tubes (4)

Loading dye

Trays for staining

Sterile water

Ziplock baggie

Marking pen

400 mL beaker for buffer

Container for waste

Beaker or graduated cylinder for agarose

Mini ice bucket

Materials: (Per Class)

Water bath

Crushed ice containers

Microcentrifuges

Documentation stations:

Camera

Film

Filter

Hood

UV transilluminator

Ethidium bromide

Staining solution

[1 mg/ml]

Purpose

In this laboratory, to prepare and analyze a simulated DNA fingerprint, the student will:

Cut DNA samples – the evidence! – by incubating it with a restriction enzyme.

Load an agarose gel with the restriction digest,

Conduct gel electrophoresis to spread out the mixture of DNA fragments in the digest,

Stain and photograph the gel to visualize the DNA,

Analyze the resulting banding pattern or “DNA fingerprint,” and use it to “solve” an imaginary crime.

STUDENT COPY:

Restricting DNA samples (“Crime scene” or “suspect”)

Each team should obtain 4 reaction tubes.

Label them:

CS (DNA from the crime scene, will be cut with enzyme)

(DNA from suspect #1, will be cut with enzyme)

(DNA from suspect #2, will be cut with enzyme)

(DNA from suspect #3, will be cut with enzyme)

Obtain DNA, enzyme and necessary reagents as directed by your teacher. Keep the DNA, buffer, and enzyme ON ICE. Add the appropriate solutions in order from top down as indicated below: (Loading matrix)

| |DW |DNA |5X R Buffer |En2 |

|CS |4µl |2µl |2µl |2µl |

|1 |4µl |2µl |2µl |2µl |

|2 |4µl |2µl |2µl |2µl |

|3 |4µl |2µl |2µl |2µl |

After the three solutions have been added to each tube, closet he caps tightly and mix by giving the tubes a quick 2 – 3 second spin in a microcentrifuge (Be sure tubes are balanced in the rotor)

Incubate at 37oC for at least 60 minutes. Let stand overnight at room temperature or refrigerated temperature.

Cast an agarose gel

Prepare your casting tray (gates up) and insert a 12-well comb in the end slots.

Obtain a beaker with 30 mls of liquid agarose, which has been kept at a 65-70 degrees C in a water bath. Pour the agarose evenly into the casting tray.

DO NOT JAR OR MOVE the casting tray as the gel solidifies. This ensures a smooth, even gel. As the agarose polymerizes (about 10 minutes), it changes from clear to slightly opaque.

While you are waiting, fill the plastic electrophoresis box with about 800 mls of TAE electrophoresis running buffer.

When the gel has solidified, lower the “gates” on the casting tray and submerge the casting tray onto its platform in the gel box. The comb should be located at the cathode end (Black lead; (-) end). The level of the buffer should be only a few mm above the surface of the gel. Add more buffer if needed.

Carefully remove the comb from the gel (pull it straight out). You’ll notice that this left behind 12 little empty “slots” or wells in the gel.

Load an agarose gel

To each of your four tubes, add 1 µl loading dye. Then give them all a quick spin in the microcentrifuge to mix the dye. (Be sure to BALANCE the tubes in the rotor!)

Is your gel ready to load? It should be in the gel box, under buffer solution; the comb should have been removed and the 12 empty wells in the gel should be at the cathode end of the box. You have four samples to load.

By convention, DNA gels are read from left to right, with the wells located at the top of the gel. With your gel lined up in its box with the wells to you rleft, the contents of Tube “1” should be loaded in the well closest to you. Thus, when the gel is turned so that the wells are at the top, “1” will be in the left-hand corner.

Fill the pipet tip correctly to 11 µl. Position your arm and pipet over the gel box. Steady that arm with your other arm. Lower the pipet tip under the surface of the buffer to the top edge of a well. Load all 141 µl of each sample into a separate well in the gel.

BE VERY CAREFUL NOT TO PUNCTURE THE BOTTOM OF THE GEL

Gently depress the pipet plunger and slowly expel a sample into the well. Keep the plunger depressed while removing the pipet tip out of the buffer.

Change tips between samples.

THE GOOEY DEVELOPING FLUID ON THE BACKING IS CAUSTIC – DO NOT LET IT TOUCH YOU.

Camera Operation:

Lift the lid of the transilluminator and replace with the camera. Wait 5 seconds for the UV light source to come on underneath.

Select aperture and shutter speed: try f 4.5, 1 sec. to start.

Look for a white film tab (this says there is film in the camera)

Steady the camera and squeeze the cable release to take the picture.

Grasp and pull out the white tab. Then pull out the yellow tab, which is actually on one end of the photograph, right out of the camera. This starts development. After 45 sec., separate picture from backing (negative).

Part IV. Gel electrophoresis

The term ‘electrophoresis’ literally means “to carry with electricity.” It is a technique for separating and analyzing mixtures of charged molecules. Due to its sugar-phosphate backbones, DNA is a negatively-charged molecule. When placed in an electric field, it will migrate toward the anode (+). The speed of migration of DNA in an agarose gel depends on the size of the piece; small pieces experience less resistance and move faster (farther) than the large pieces.

CATUIONS

|POWER SUPPLY |

If two teams are connecting their gel boxes to one power supply, be sure to communicate with each other whenever the power supply is turned ON or OFF.

With the power supply off, secure the lid of the gel box and connect the leads to the same channel of the power supply (Red-red, black-black).

Turn the voltage knob down to ZERO. Then turn the power supply ON. Set the power supply to about 100 Volts.

Notice there is a switch to direct the LED display to read in either volts or milliAmps. Use it to verify how much current is flowing through the gel. (It should read about 40 milliAmps with one gel box hooked up or 80 milliAmps with two boxes hooked up with 1X TAE buffer.)

Shortly after the voltage is applied, you should notice something happening to each electrode… what is it? Allow electrophoresis to proceed until the dye, and DNA, are out of the wells and safely into the agarose matrix.

Turn the power supply OFF and disconnect the leads.

Remove the casting tray from the gel box. CAREFULLY slide your gel off the casting tray and into an empty plastic tray or “boat.” Label the rim of the tray with your initials. Place the gel and “boat” into a ziplock baggie and seal. Your teacher will instruct you how to store the gels overnight.

Day 3

TO RESUME ELECTROPHORESIS, CAREFULLY slide your gel back onto a casting tray. BE SURE YOUR DNA WILL BE GOING IN THE CORRECT DIRECTION! Be sure the casting tray’s gates have been lowered. Set the tray into a gel box containing the buffer; connect the gel box to the power supply; turn the power supply ON and adjust the voltage as instructed above.

Continue to electrophorese until the fastest-moving dye front has advanced at least 2/3 to ¾ of the way across the gel (about 45 minutes time).

Then, turn the power supply OFF and disconnect the leads.

Remove the casting tray from the gel box. CAREFULLY slide your gel off the casting tray and into its labeled plastic tray or “boat”.

Day 4

Part V. Stain and photograph the Gel

There are many stains and tagged probes that allow us to visualize DNA. In our lab, we will stain the DNA with a fluorescent dye called ethidium bromide (EBr). EBr slides between the runes of the DNA double helix. When excited by ultraviolet (UV) light, the EBr absorbed some of the energy and emits orange (visible) light.

CAUTIONS:

Do not allow your skin, eyes, mouth, etc. to come in contact with ethidium bromide solution. Always wear goggles and gloves if working with this chemical. If you accidentally spill ethidium bromide staining solution clean it up appropriately.

UV light can damage unprotected eyes and skin. Never look directly into an unshielded UV light source.

FOTODYNE transilluminators are safe, since they will not turn on unless the acrylic safety shield is lowered over the gel.

Bring your tray to the Staining Station

An operator wearing gloves (probably your teacher), will transfer your gel to a staining dish. The staining dishes contain a dilute EBr solution.

The gel will be left in the EBr stain for 20 minutes, and then rinsed in distilled water for 5 – 10 minutes (to increase contrast and make the gels safer to handle).

Your teacher will put your gel on the surface of the UV transilluminator. When the safety-lid is closed, this “box” emits ultraviolet light. The ultraviolet light makes EBr-coated DNA fragments glow.

The teacher may use one of several methods to record your results. If s/he takes a Polaroid 667 photograph of your gel, after about 45 sec. of developing time, peel the backing away to separate the print from the negative.

Upon completion of this lab:

Dispose of designated materials in the appropriate places.

Leave equipment as you found it.

Check that your workstation is in order.

Wash your hands.

DNA FINGERPRINTING SIMULATION

Sample Crime Scenario

Students Copy!

A jewelry store in San Mateo was robbed last night! The perpetrator appeared to cut him/herself on the broken store window; blood was found on glass at the scene of the crime. The blood-spattered shards of glass were taken to a forensics lab to extract DNA.

Teacher’s Copy!

I read the above info out loud, but did not allow students to take notes (just as most jury members are not allowed to take notes during a trial). Later, based on students’ reports, I noticed a lot of erroneous recollections! Some students told me the busboy had a bag with him; that the jewelry store owner was a man; that the busboy was in fact a bellboy from a local hotel; and all the alibi’s and motives were mixed up!

Students Copy:

|SUSPECTS |Woman, in 40’s |Man, ex-con, in 30’s |Young man in 20’s |

|ACTIVITY AT TIME OF ROBBERY |Owner of jewelry store. Had worked |Notorious jewel thief paroled from |Busboy at restaurant near jewelry |

|(probable cause) |in the store that day. Was |prison. Crime statistics show that|store. Was seen running down the |

| |observed meeting with jewelry |a large fraction of ex-cons return |street near the store late at night|

| |appraiser in “bad” part of town |to criminal behavior. Police had |(after midnight). |

| |earlier. |observed the man in the area of the| |

| | |jewelry store. | |

|MOTIVE |Would collect on theft insurance; |Had returned to theft/fencing of |Was in need of money to support his|

| |if she had “arranged” for the |jewelry for money |family; his hours at the restaurant|

| |robbery, she’d also still have the | |had been cut way back. |

| |jewels. | | |

|ALIBI |Had gone home from the store not |Had gone to a crowded movie by |Had worked the late shift and was |

| |feeling well. Spent evening alone |himself. Ticket taker could |running down the street to catch |

| |at home. As far as the appraiser –|neither confirm nor deny that the |the bus. |

| |he was a friend of a friend. |man had indeed gone to that | |

| | |theater. | |

Images of DNA;









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