Every living organism carries within it a #NUCLEIC ACID POLYMER (NAP) that contains a CODED MESSAGE with instructions on how to make more of the SAME organism. This NAP is an organism's GENOME. The genome contains all the GENES an organism needs to metabolize, grow, and reproduce. In MOST organisms this genomic material is DNA. Some viruses, but NO HIGHER LIFE FORMS, use RNA polymers as genomic material. To study the genes of any organism it is necessary to PURIFY its genome and to isolate the individual genes therein. In the last 25 years techniques for CUTTING OUT individual genes from genomic DNA, and then CLONING and AMPLIFYING them have made the molecular biology revolution possible. The crucial reagents in this process include RESTRICTION ENZYMES, that cleave DNA only at specific sequences located RANDOMLY along DNA strands. Because of this randomness a cell's genome may contain, for a given restriction enzyme, 0 to thousands of cleavage sites. Thus, when a DNA sample is treated with a particular restriction enzyme the number of fragments produced equals (for a linear DNA strand) the number of unique cleavage sites + one. Because of the random placement of the sites, the lengths of the DNA fragments vary from a few to a several 1000 or more base-pairs. DNA-fragments of a 1000 or more base-pairs often contain one or more genes. Further, the pattern of fragments produced by treating genomic DNA from each organism is a constant for each restriction enzyme and as such can be used as a FINGERPRINT to identify that organism. That is, another member of that species is likely, because of mutations, to have one or more different sites for the same restriction enzyme.
Figure 1. The DNA chain on the left shows the A-T and G-C complementary base pairing. On the right is a #southern blot of DNA fragments of genomic DNA separated on an agarose gel and hybridized with a probe that lights up only the DNA fragments the probe binds to because of complementarity.
In this exercise we will demonstrate how one important molecular biological technique, GEL ELECTROPHORESIS SEPARATION OF MOLECULES, is performed. We will use dyes rather than DNA as they are visible and less toxic than some of the reagents that are used in viewing DNA fragments, plus DNA samples are expensive and difficult to obtain for mass use. However, the principles involved in separating the dye molecules are the same as for separating DNA molecules. Briefly, when charged molecules are placed between a positive and negative electrode, the positively charged molecules move towards the negative electrode and the negatively charged molecules migrate towards the positive electrode (opposites attract). A dramatic example of this phenomenon (charge migration) is that of lightening. Since molecules free in solution easily disperse, agarose is used to restrain the diffusion of the molecules being separated. The agarose contains large pores (empty spaces) through which charged molecules can move while being restrained in a limited area. Thus DNA or dye molecules, dissolved in a dense solution, sink into wells formed in the agarose. When electrical current is applied, the molecules in the wells migrate through the agarose gel towards the respective electrodes while remaining in a relatively compact band. The smaller the molecule the EASIER it is able to move through the pores and thus the more RAPIDLY it migrates. This results in a graded (proportional) separation of molecules of different sizes from the well towards the electrode; with the largest molecules moving the shortest distance from the wells and the smaller molecules moving the farther distance.
Draw the relative position of the various dyes on the gel, labeling each with its color, mm traveled from the well and its charge.
SAMPLE QUESTIONS: You should be able to answer
these questions at the conclusion of this laboratory. Fingerprints and blood typing as means of identification have been around for several generations. Both have been presented in numerous movies, plays and books as techniques for solving crimes and identifying criminals. Both have entered that realm of "COMMON KNOWLEDGE" and are universally accepted. In the case of fingerprints, anyone can compare their own fingers with those of others and plainly see that all fingerprints are unique. Further, everyone with a minimal education and intelligence can smear a bit of ink on their fingers, press it onto a piece of paper and immediately understand how this data could be collected and analyzed. Finally, law enforcement personnel, can, with minimal training and experience, quickly grasp the means of accurately collecting this data and its analysis is straightforward; i.e., it involves minimal STATISTICS, of which most people are skeptical. Further, it doesn't require an expensive and complex molecular biology laboratory or suffer from an obtuse vocabulary of scientific terminology and arcane concepts. With blood typing almost everyone understands its medical significance (different people have different types) even if they don't know the science behind it.
But this is not the case with DNA fingerprinting. The use of DNA as evidence in criminal and civil law cases exploded on the justice system about 15 years ago. In the current environment of scientific illiteracy, the description of DNA fingerprinting, with its unfamiliar terminology and concepts of DNA, genetics, evolution, restriction enzymes, Southern blots, probes, electrophoresis and DNA-fragment patterns, and complex statistical analyses, one can understand why this technology confuses and frustrates most nonscientists. Although the legal system and law enforcement authorities have, in most cases, grasp the usefulness of this technology, average citizens, >50% of whom do not even know what DNA is or its role in genetics, remain woefully ignorant about it and, like all ignorant people, are suspicious and dismissive of what they don't understand.
The law has suddenly gained a powerful new technique for use in solving criminal and civil cases and a race is ensuing to implement it. However, the rapidity with which DNA fingerprinting has moved from the experimental laboratory into the law courts has not given the public time to understand its value and limitations. The O.J. Trial spotlighted these problems. Also some components of the judicial system do not understand how this data should be used or exactly what it means in various circumstances. This has resulted in DNA evidence being used inappropriately and/or presented in a confusing manner. For example, the post-trial interviews of the O.J. jurors indicate that most of them simply disregarded the DNA evidence because they could not understand its significance. Also, it was equally clear that the lawyers and their "DNA expert witnesses" (usually scientists) did not know how to present this information to the O.J. jury in an understandable way (They "turned off" the jurors).
In this film you will see how DNA-fingerprinting can be employed to both convict the guilty and to free the innocent. In the latter case, a significant number of men convicted for rape have had their verdicts reversed in the last few years based on DNA evidence obtained from rape-victims' semen-contaminated clothing, when it has been shown that the DNA in the semen does not match that of the convicted person. As discussed in the NetText101, the quality of the testing is crucial in demonstrating the validity of the DNA-fingerprinting results. If proper controls are not run, if the samples are allowed to become contaminated or degraded (or tampered with), if the personnel carrying out the tests and their interpretation are not adequately trained professionals or if record-keeping is not of the highest quality, the data must be suspect.
In this video you will see the results of the separation of different sizes of DNA fragments in agarose gels as was done in Part A of this exercise. Each set of DNA fragments was produced by cutting the DNA with one or more restriction enzymes which produces a large range of sizes of DNA fragments that are detected by hybridization with labeled #PROBES. Since the number and location on DNA of restriction sites differs depending on each person's alleles and mutations, differences in DNA restriction banding patterns can always be detected if samples are cut with several different restriction enzymes.
Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for
commercial purposes.
SCIENCE HALL, ROOM 440CA
PHONE: 509-335-5108
E-mail address: hurlbert@wsu.edu or hurlbert@pullman.com
OFFICE HOURS: Mon. Wed. 2-4 PM.
