DNA & Biotechnology Lab Learning Objectives: Students should be able to: 9.1 DNA Structure and Replication 1. Explain how the structure of DNA facilitates replication. 2. Explain why DNA replication is semi-conservative. 9. 2 RNA Structure 1. List the ways in which RNA structure differs from DNA structure. 9.3 DNA and Protein synthesis 1. Define and describe the processes of transcription and translation during protein synthesis. 2. State the roles of DNA, mRNA, tRNA and amino acids in protein synthesis. 9.4 Isolation of DNA 1. Understand the importance of DNA technology. 2. Describe the isolation of whole genome DNA. 9.5 DNA fingerprinting 1. Explain the steps to create a DNA fingerprint. 2. Analyze a fingerprint. Task 1: DNA Structure and Replication 1. Read “Introduction” (pg 227) and section 17.1 “DNA Structure and Replication” (pg 228). 2. Complete Figure 17.1 “Overview of DNA structure” (pg 228) 3. Read and complete “Observation: DNA Structure”, questions 3-7 (pg 229). In Table 17.1 “Base Colors” skip the “in your kit” section. 4. Read and complete “Observation: DNA Replication”, questions 1-5 (pgs 230-231), and Table 17.2 “DNA Replication” (pg 230). Do not continue until your TA checks your progress. Task 2: RNA Structure 1. Complete Figure 17.3 “Overview of RNA Structure” (pg 231), as directed in “Observation: RNA Structure”. Answer questions 1-3 (pg 231) and complete Table 17.3 “DNA and RNA Bases” (pg 231).2. Complete Table 17.5 “DNA Structure Compares with RNA Structure” (pg 232). Do not continue until your TA checks your progress. Task 3: DNA and Protein Synthesis 1. Read section 17.3 "DNA and Protein Synthesis" (pg. 232) and “Transcription” (pg 233). Complete Figure 17.4 “Messenger RNA (mRNA)” and Table 17.6 “Transcription” (pg 233). 2. Read “Translation” (pg 234). Perform “Observation: Translation”, answer question 1-2 and complete Figure 17.6 “Transfer RNA diversity” (pg 234). 3. Complete Table 17.7 “Translation” and Figure 17.7 “Protein synthesis” (pg 235). 4. Read section 17.4 “Isolation of DNA” (pg 237). Perform Experimental Procedure: “Isolation of DNA” following this procedure (Note: Do not follow the manual’s instructions): 1. Cut the onion into several small pieces and place in the mortar. 2. Ad 10ml of detergent solution. 3. Gently grind the onion with the pestle until you obtain a mushy mixture. 4. Place a piece of cheesecloth in the funnel and filter the onion mixture into a beaker. 5. Decant as much of the onion mixture as possible into the beaker, trying to leave most of the foam behind. 6. Add 3ml of the protease solution to the beaker containing the clean, filtered onion solution. Swirl gently to mix. 7. Slowly and gently add 3-5ml of ice cold ethanol to the onion/protease solution. Disperse it slowly against the side of the beaker so that it flows over the surface of the solution. The purpose is to maintain two separates layers. If the layers mix you will not get a good DNA isolation. 8. Let the solution sit for about 3 minutes. Don not disturb it in any way. You should see the DNA begin to precipitate into the alcohol layer. 9. Collect the DNA by gently spooling them onto the glass Pasteur pipette.4. Answer question 6 (pg 237). Do not continue until your TA checks your progress. Task 4: DNA Fingerprinting (Adopted from Sylvia S. Mader. 2008. DNA and Biotechnology. In: Human Biology Laboratory Manual, 10th ed. Boston: McGraw-Hill). 1. DNA Fingerprinting: A genome is all the genetic material in a set of chromosomes. The genome contains portions of DNA (i.e. genes) that code for the various proteins and specialized RNAs (tRNAs and rRNAs). Other DNA portions are sometimes called “junk” because they do not code for proteins or RNAs (these sections are repeats of the same short sequence of bases over and over again). Detectable differences in the noncoding portions of the genome comprise an individual’s DNA fingerprint. DNA fingerprinting has many uses. DNA fingerprinting is used by (1) police and courts to identify a person who has committed a crime; (2) genetic counselors to determine if an individual has or will develop a genetic disorder; (3) lawyers to determine relatedness between individuals; (4) scientists to determine the identity of individuals based on minimal remains after death. DNA fingerprinting is also used by (1) conservation biologists to determine the genetic dissimilarity of males versus females for breeding purposes; (2) evolutionary biologists to construct evolutionary trees; and (3) taxonomist to distinguish species. DNA fingerprinting requires three steps (Figure 1): 1. Fragmentation of a selected portion of the genome. The DNA is digested with restriction enzymes, which results in variable sized fragments unique to the individual. 2. Gel electrophoresis. The DNA fragments are separated according to their length, and the result is a DNA pattern unique to the individual. 3. Analysis of the DNA pattern. The pattern is revealed by using radioactive probes or by staining. If you are dealing with just a small portion of the genome, staining of the gel is sufficient to reveal the fragment pattern.Figure 1. DNA Fingerprinting. During DNA fingerprinting, DNA samples are digested to fragments. The fragments are separated by gel electrophoresis, and then the resulting fragment length pattern is observed. In this example, the sample in well I could be from the crime scene, making the DNA in well II from the criminal; or the DNA from well I could be from a parent , making the donor of the sample en well II his child; or sample I could be from a body part, making sample II the deceased individual. In the last case, sample II would have been taken from some object known to belong to the individual in question. 2. Analyzing the DNA pattern: Figure 2 shows the fragment patterns of 4 samples: one collected from a crime scene (CS) and three suspects (S1, S2, S3). Which of the suspect’s DNA matches the DNA sample collected from the crime scene?