07 - Lion Family Reunion: Conservation Biology Genetics
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS Several methods exist that allow scientists to read DNA and visually see areas that are variable within a population or a species. One popular method is restriction fragment length polymorphisms (RFLP) analysis. This technique allows scientists to visualize small nucleotide changes in the DNA of different samples as difference in the number and lengths of bands produced when extracted DNA samples are digested by restriction enzymes and then separated by agarose gel electrophoresis. Restriction enzymes are proteins that cut DNA molecules at very specific nucleotide sequences known as recognition sites. For example, the popular restriction enzyme EcoRI cuts DNA whenever the sequence of nucleotides is GAATTC. A sample digested with EcoRI will produce several fragments of DNA of different sizes depending on how many times this recognition site is present in the DNA and where these recogni- tion sites are located (Figure 4). Because the presence or absence of a fragment is directly related to a change in the nucleotide sequence, RFLP patterns contain both diversity and evolutionary information. After a sample has been digested by a restriction enzyme the number of fragments produced by this digestion and the size of these fragments can be determined using agarose gel electrophoresis. First samples of DNA are loaded into wells within an agarose gel. The gel is then placed in an electrophoresis chamber containing a buffer solution and electrodes. Next a direct current is applied from a power source. Since DNA is negatively charged in the neutral buffer it migrates through the gel towards the positive electrode. During this process, the agarose gel - which consists of microscopic pores - acts as a molecular sieve and separates the different DNA fragments according to their size. Shorter fragments migrate quickly through the gel and will travel farther down the gel during the experiment than longer fragments that move more slowly through the gel. Following electrophoresis the RFLPs are “scored” by recording all the fragment lengths generated by all
the samples in a study and then by identifying whether each individual has (0) or lacks (1) that particular fragment. This creates long sequences of 0s and 1s which are then used to determine the number of unique haplo- types and the haplotype of each individual. In phylogeography, multiple enzymes are often used to increase the number of data points in a study. This is important as larger datasets create more reliable and more descriptive phylogenies and haplotype maps as well as more precise predictions about ancestry and historical events. In this experiment, you are a conservation biologist who is working with two adolescent lion cubs the were rescued from a private house. Based on their age, you’ve decided to reintroduce them into the wild (or wildlife sanctuary) rather than to a zoo. Using their genetic profiles and information about lion phylogeography, you will identify the best sanctuary for them. In Modules I and II you will run gel electrophoresis to determine the lions’ restriction fragment length polymorphisms (RFLP), and in Module III you will determine the haplotype of each lion. Using this informa- tion, you’ll determine where to reintroduce each animal so that they’re optimally matched to their environment.
AATTC G
G CTTAA
PLASMID A
PLASMID B
Plasmid A
Plasmid B
Figure 4: Restriction Enzyme Digests to Band Pattern.
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