LABORATORY EIGHT

DNA FINGERPRINTING

OVERVIEW:

            Methods for identifying individual living things, including humans, have always been of great interest.  In order to make an individual identification system work, we must discover some attribute of the organism that is unique for each and every genetically distinct individual or person.  For example, the identification of individual humans in criminal cases has historically relied on fingerprints, and is based on the knowledge that with the exception of identical twins, no two human beings have the same pattern of ridges and grooves on their fingertips.  But, as you might suspect, many cases arise in which the elements of this system, decipherable fingerprints, are not available.  The ideal system would be one that could identify an individual from very small samples of the individual's tissues, such as hair shafts, fingernails, a few blood cells, or samples of body fluids.

OBJECTIVES OF LABORATORY:

•To learn the biological technique of gel electrophoresis.

•To understand the principles behind DNA fingerprints.

•To make and analyze a set of DNA fingerprints.

INTRODUCTION:

            You are learning from your studies of mitosis, meiosis and Mendelian genetics that it is so unlikely that any two individuals will be genetically identical (again, identical twins, two individuals resulting from a single fertilization, are an exception) that we can state with confidence that the hereditary material of every human is unique.  If there were some way to detect that uniqueness, we would have a method for individually identifying nearly every human alive!  The technology of molecular biology has provided us with such a system, called DNA fingerprinting, or typing.

            The uniqueness of each individual's genetic makeup has its foundation in the molecule Deoxyribose Nucleic Acid, or DNA, which composes the genes.  DNA consists of sequences of subunits called nucleotides arranged in long, linear chains.  A pair of such chains makes up each DNA molecule; the molecules may be millions of bases long.  While there are only four different nucleotides, the large number and arrangement of them in each DNA molecule ensure that there will be more than enough complexity to allow each of billions of humans to have distinct sequences.  Thus, if we have the sequence of an individual on file and can match it to DNA found at, for example, a crime scene, this makes it almost certain that that individual was present at the scene.

            Early in the development of molecular genetics, it was discovered that many micro-organisms, especially bacteria, have restriction enzymes, which can digest, or break up, DNA molecules.  These enzymes are part of the bacterial cell's defense against viruses; by cutting up the viral DNA, they protect themselves from infection.  The enzymes do so by "recognizing" certain short sequences of bases in DNA, called recognition or restriction sites.  Whenever an enzyme molecule locates one of these sites, it cuts the DNA at or near that point.  About 1500 different restriction enzymes have been found, and each of them has its own recognition site.  Therefore, each restriction enzyme cuts DNA at a different place.

            If we expose DNA to a restriction enzyme, it will be cut into a number of fragments.  The number and size of the fragments depends on where, and how many times, the recognition site is found in that particular DNA.  With the millions of nucleotides in each DNA molecule, it is not surprising that the distribution and number of specific recognition sites is unique or very nearly unique in every individual's DNA.  Therefore, the fragments resulting from such a treatment will be themselves unique in number and size for each individual.  And even if two individuals were closely enough related so that they could produce nearly identical arrays of fragments with one enzyme, there are 1499 others to try—eventually an enzyme, or combination of enzymes, will reveal the inevitable differences.

            Let's walk through a hypothetical case.  Imagine that a female student from another college was assaulted while visiting Hampden-Sydney.  The description she gave police of her attacker was vague; it fit about a dozen HSC students, three of whom were known to have been in the vicinity of the crime scene at the time.  The victim was able to scratch her attacker's hand, and tiny bits of skin were removed from under her nails.  DNA was extracted from these bits, and through a process called polymerase chain reaction, or PCR, the tiny sample of DNA was amplified many times, providing enough for testing with restriction enzymes.  After exposure to the enzymes, another technique called gel electrophoresis (which you'll be doing in this lab) is used to separate the fragments according to size.  The technique is based on the fact that DNA carries a slight negative charge and will be attracted to the positive pole in an electrical field.  Smaller pieces will travel faster because they can negotiate the pores in the gel with more ease.  When the current is turned off, the fragments will be strung out along the gel in order of size.  The result is a pattern on a small slab of gel that will be unique to this DNA—this is itself the DNA fingerprint.  Of course, the fingerprint is useless unless it can be matched to a known fingerprint.  Therefore all of the suspects must agree to submit a small quantity of blood, or hair or skin, that will undergo the same procedures.  We hasten to add that no one in the United States can be compelled to provide the blood sample; it must be done voluntarily, at least for now.  If the fingerprint derived from any of the blood samples matches that from the skin fragments, the attacker has been identified.  Of course, our legal system requires that matters of guilt and innocence be determined by a jury, and not by a laboratory test.  DNA fingerprinting only provides evidence for the jury's consideration.

            Another situation in which DNA fingerprinting has proven valuable has been in paternity suits.  The mother of a child may be able to use this technology to prove the identity of the father of the child from among an array of potential parents.  Similarly, refugee children can be reunited with their biological parents even though many years have passed since their separation, and they may find one another mutually unrecognizable visually.  Remember that a child inherits half of its genetic information, and thus half of its DNA, from each of its parents. Thus the recognition site patterns of the child will be a mixture of those of both its parents, but will not resemble particularly closely those of unrelated individuals.  In other words, all of the DNA fragments in the fingerprint of the child that do not match the mother's fingerprint will match the father's.  In this way an almost unequivocal identification can be made.  But please remember that such suits are settled by jury trial or by a judge, and that DNA fingerprinting is only submitted as evidence.

            In this laboratory you will study the technique of DNA fingerprinting through a simulation—that is, no real human DNA will be used, but the fingerprints you will make are genuine and will closely resemble the real thing.  The analysis of the "fingerprints" will be up to you!

PROCEDURE:

1.         Your instructor will briefly explain the structure of the DNA molecule, the function of restriction enzymes, and how DNA fingerprinting works.

2.         Your instructor will explain and demonstrate the gel electrophoresis apparatus, and show you how to prepare a gel.

3.         Each group will be provided with five small tubes containing DNA samples, as follows:

                        Tube                            Contents

                        A                                 Standard DNA fragments

                        B                                  Mother's DNA cut with enzyme

                        C                                 Child's DNA cut with enzyme

                        D                                 "Father" 1 DNA cut with enzyme

                        E                                  "Father" 2 DNA cut with enzyme

Obtain your samples labeled A-E and heat them in a water bath at 65º C for 2 minutes. 

4.         Read the Protocol for Using Pipettes, which is located after the Assignment at the end of the lab, and practice using the pipette before proceeding with the next step.

5.         When your gel is ready (consult your instructor to make sure), load the DNA samples into the wells in the gel.  Be sure that the entire contents of each sample tube gets into the well.  Number the wells from left to right, so that well #1 corresponds to sample A, and so on.  The DNA fragments will migrate from the wells toward the positive pole of the apparatus, along lanes that correspond to the positions of the wells.

6.         Close the cover of the electrophoresis apparatus.  Be sure that the negative and positive indicators on the cover are in the right orientation.

7.         Connect the black wire to the black output of the power source, this is the negative pole, and the red wire into the red output of the power source, the positive pole.

8.         Set the voltage on the power source according to your instructor's directions and turn it on.  Check the electrodes.  If tiny bubbles are forming on them, the current is flowing.

9.         Allow the electrophoresis to run for the time prescribed by your instructor.  This will be from 45 minutes to an hour.

10.       Remove a small slice of the gel from the upper right corner, the right corner closest to the negative electrode, so that you will be able to keep the gel in correct orientation.

11.       Remove the gel on its bed.  Keep one hand on each end of the bed so that the gel will not slip off.

12.       Follow the instructions for DNA Blue InstaStain included here. 

13.       To analyze the fingerprints, match the bands in the mother's lane to those in the child's.  Any unmatched bands should match those in one of the potential fathers' fingerprints.

Assignment:

Your instructor will assign some or all of these questions for you to analyze.

1.         Gregor, Zelda and Ignatz were separated from their parents as infants when Bork and Quanta Globitz were imprisoned by the cruel regime of General Yech.  Twenty years of turmoil in Ruritania has caused the loss of all records that could be used to bring parents and children together.  Would it be possible to reunite these young adults with their biological parents?  Explain in detail, using a narrative.

2.         Could DNA fingerprinting be used to determine if two people are siblings?  Explain.

3.         Virginia's central forensic laboratory has in storage blood and tissue samples collected from more than 100,000 convicted felons.  The Chief Pathologist estimates that it would cost about two million dollars to prepare DNA fingerprints for all of them.  Would this be a wise expenditure?  Explain.

4.         Persons now placed under arrest are compelled to be fingerprinted.  Do you think that suspects in criminal cases should be compelled to give blood samples for DNA fingerprinting?  Explain your view.

5.         Your lecture instructor may have explained how blood typing can be used in paternity cases (if you haven't studied this, ask your laboratory instructor about it).  How does DNA fingerprinting represent an improvement over that method?

6.         Read Lab 8, Natural Selection and Gene Frequencies.

Protocol for Using Pipettes

Note: DO NOT attempt to adjust the volume by turning the cylinder (control button) on top of the pipette. These instruments are delicate and expensive. Instead, press gently down on the control button and notice that there are two “stop” points; the second is reached by gently pressing through the first stop.

To withdraw and dispense liquids:

1.         Attach a tip to the pipette without touching the tip with your hands. To do this, press the

narrow end of the pipette directly into a tip as it sits in the box. This avoids contaminating the tips with material from your fingers. A gentle, even pressure is all that is necessary to securely attach the tip. Lift the tip from the box with the pipette.

2.         Press the control button down to the first stop and hold.

3.         Hold the pipette vertically and immerse the tip approximately 3 mm into the liquid.

4.         Let the control button glide back slowly and smoothly.

5.         Slide the tip out of the liquid along the inside wall of the vessel.

6.         To dispense the liquid, hold the tip at an angle against the inside wall of the receiving vessel or gel well.

7.         Press the control button slowly down to the first stop and wait approximately 2 seconds.

8.         Press the control button down to the second stop to empty the tip completely.

9.         Continue to hold down the control button and slowly slide the tip out along the inside wall of the vessel.

10.       Let the control button glide back slowly. Eject the tip by pressing the control button to the final stop, which is beyond the second stop.