Genetics 2Biology 3 - LA Mission CollegeGenetics 2A - DNA Fingerprinting Using Gel ElectrophoresisGenetics 2B - Punnett Square Problems1Genetics 2A - DNA Fingerprinting: Separating DNA Molecules Using Gel ElectrophoresisObjectives! understand how restriction enzymes can be used to study differences in the DNA of organisms! understand how different sizes of DNA molecules can be separated by Gel ElectrophoresisBackground InformationIn genetics, we are interested in examining whether the segments of DNA on a chromosome are similar or differentbetween individuals. One way of examining the similarities or differences between the DNA of two or moreorganisms is to use restriction enzymes to cut the DNA into fragments.Restriction enzymes are ADNA scissors@ that cut the double helix molecule at very specific nucleotide sequences.These enzymes, which are generally isolated from bacteria, will cut the double stranded DNA molecule atsequences which are specific for each enzyme. Here are examples of some restriction enzymes and the sites atwhich they will cleave:Bam HI 5'-GGATCC-3' Hae III 5'-GGCC-3'3'-CCTAGG-5' 3'-CCGG-5'Pst I 5'-CTGCAG-3' Hinf I 5'-GATC-3'3'-GACGTC-5' 3'-CTAG-5'One interesting feature of mammalian DNA (humans included!) is the existence of numerous short repeatingsequences of nucleotides that can exist on either side of a gene of interest. These short repeating sequences can befew, or many, depending on the individual that is studied. It is for this reason that we call these small stretches ofnucleotides variable number tandem repeats (VNTR=s). (See Figure 1)Genotype #1 RS TGTTA TGTTA TGTTA TGTTA----------------GENE X--------------------TGTTA TGTTA TGTTA TGTTA TGTTA RSGenotype #2 RS TGTTA TGTTA----------------GENE X---------------------TGTTA TGTTA TGTTA RSGenotype #3 RS TGTTA TGTTA-----------------GENE X--------------------TGTTA RSFigure 1 - Examples of three possible genotypes of VNTR=s around Gene X (VNTR is TGTTA)As shown in the figure above, the genotype of an individual can be determined by cutting the DNA with arestriction enzyme at the restriction site (RS) that is known to be associated with the gene of interest. This willresult in the production of DNA fragments of a particular length, demonstrating the specific DNA sequence nearthat gene of the individual being studied. Since for each gene that can be examined, there are numerous possiblegenotypes (many different alleles) for that segment of DNA, it is possible to use these restriction fragment lengthpolymorphisms (RFLP=s) to study the similarities and differences of the DNA between organisms.It is by using this technology that scientists can determine if one person is related to another person, or if cells left2at the scene of a crime may or may not correspond with the DNA of a suspect. The DNA from a person =s cells canbe isolated and subjected to a restriction enzyme that is associated with the production of restriction fragments thatan investigator wishes to examine.One only requires a method of observing the size of the DNA fragments that result from the use of the restrictionenzymes. This is the purpose of the technique known as Gel Electrophoresis.One of the easiest ways to separate two different molecules in a mixture is to separate them based on their size. Toseparate pieces of DNA of different sizes we use a process known as Gel Electrophoresis (Aelectric@Aseparation@). It is best to think of this process as a Amolecular race track.@ A mixture of DNA molecules isplaced in a Awell@ at the end of a rectangular gel. The well acts as a Astarting gate@ for the DNA molecules.The Arace@ is begun by creating a voltage across the surface of the gel, with one side being positive and the otherside being negative. Since DNA molecules are negatively charged, they will start to Arun@ toward the positiveside of the gel (opposites attract).As the DNA molecules in the mixture start to move, the smaller pieces of DNA will move faster than the largerpieces of DNA. Thus, we can separate different length segments of DNA by Arunning@ them on a gel. (SeeDiagram 1)Diagram 1 - Separation of DNA Molecules of Different Sizes Using Gel Electrophoresis3A. Setting Up a Gel in the Electrophoresis Box1. Examine the diagram of the Gel Electrophoresis apparatus below.B. The Case of Who Assaulted Sally JonesTwo months ago, Sally Jones was sexually assaulted on her way home from work. She immediately went to thehospital to be treated. The physicians wisely obtained a sperm sample in order to provide police with evidence ofthe assault.The police had been investigating other similar cases in the area and had recently taken into custody two suspects.Both of the suspects denied that they were involved in the assault and provided police with blood samples.You will be required to use gel electrophoresis to examine whether the DNA from Suspect 1 or Suspect 2 matchesthe DNA taken from the sperm cells collected after the attack.The samples that you have been given have been subjected to two different restriction enzymes - each enzymeallowing you to exmine a different DNA segment. Below is a list of the tubes and the samples they contain:Tube ContentsA DNA sample from sperm collected after the crime - cut with Restriction Enzyme #1B DNA sample from sperm collected after the crime - cut with Restriction Enzyme #2C DNA sample from Suspect #1 - cut with Restriction Enzyme #1D DNA sample from Suspect #1 - cut with Restriction Enzyme #2E DNA sample from Suspect #2 - cut with Restriction Enzyme #1F DNA sample from Suspect #2 - cut with Restriction Enzyme #2C. Loading and Running the DNA Samples on a Gel1. Examine the electrophoresis gel that has already been set up for you. Notice that it is a thin layer of gel that isbarely submerged in electrophoresis buffer. There should be a set of six Awells@ to load the DNAsamples.2. Do the next steps next to the Gel Electrophoresis Apparatus.3. Set your P-50 micropipettor to 40.0 l. Using a NEW TIP each time, load 40.0 l of each sample into the well that corresponds with the sample letter. Remember to hold the micropipettor with both hands and place yourelbows on the table top.4. When you are finished loading the six DNA samples on the gel, place the power unit in the apparatus, andturn on the power. Set the voltage at 80 to 90 volts and allow the samples to
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