Using the case study, write a full lab report. In the result, the picture from the gel on this lab manual can be incorporated. Discuss on if you know who is to blame for this crime and what are the evidences.

Lab 4. RFLP Analysis of DNA Restriction endonucleases (RE) are enzymes that cut DNA at a specific recognition sequence, typically 4-6 bases in length, called a restriction site. All restriction endonucleases cut DNA between the 3′ carbon and the phosphate moiety of the phosphodiester bond so that fragments produced by restriction enzyme digestion have 5′ phosphates and 3′ hydroxyls (Fig. 8.1). Restriction endonucleases are named after the bacterium they were extracted from. The capital letter of the genus is used followed by the first two or three letters of the species name (or sometimes additional letters for the strain that it came from), followed by a Roman numeral that gives relates to the number of restriction endonucleases obtained from that organism or strain (Table 8.1). For example, Eco RI was extracted from Escherichia coli strain RY13, and Hind III was the third restriction endonuclease obtained from Haemophilus influenzae strain Rd. Sometimes the same recognition sequence is seen from restriction enzymes from different sources. For example, Hae III (GG↑CC) and Bse I (GG↑CC) both have the same recognition sequence. They are called isoschizomers. Sometimes a restriction endonuclease will recognize and cut more than one sequence. For example, Ava I (C↑PyCGPu↓G) has four recognition sequences (C↑CCGA↓G, C↑CCGG↓G, C↑TCGA↓G, C↑TCGG↓G). If another restriction endonuclease cuts any one of the sequences from these multiple recognition sequence restriction endonucleases they are called semi-isoschizomers. For example, the recognition sequence of Sma I (CCC↑GGG) is identical to one of those recognized by Ava I and the recognition sequence of Xho I (C↑TCGA↓G) is identical to another of the recognition sequences recognized by Ava I. Thus, Sma I and Xho I are both semi-isoschizomers of Ava I. Blunt ends or flush ends: It occurs when restriction endonuclease makes a simple double-strand cut in the middle of the recognition sequence. -N-N-A-G-C-T-N-N- -N-N-A-G C-T-N-N- -N-N-T-C-G-A-N-N- -N-N-T-C G-A-N-N Sticky ends or cohesive end: The two DNA strands are not cut at exactly the same position so that the resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky ends as base pairing between them can stick the DNA molecule. -N-N-G-A-A-T-T-C-N-N- -N-N-G A-A-T-T-C-N-N- -N-N-C-T-T-A-A-G-N-N- -N-N-C-T-T-A-A G-N-N The definition of a unit of a restriction endonuclease is “the amount required to digest 1 ug of DNAto completion in 1 hour at recommended temperature and buffer conditions in a 20 µL reaction”. See pages 172-176 of lecture textbook Chapter 8, plus Tables 8.1 and 8.2, and Figs. 8.1, 8.2, and 8.3. Changes in number of fragments – sequence realignments, additions or deletions of DNA, base substitutions within the cleavage sites. Fragments separated by gel electrophoresis (agarose or polyacrylamide) (Fig 8.14 iGenetics second Edition, Fig. 8.8 3rd Edition), distance moved is proportional to logarithm of molecular weight. Fragments of known size are used as internal standards (Fig. 15.2 from Lab book). See also the figure “DNA fragments separated by electrophoresis and visualized under UV light” on page 248. Steps in analysis: (1) select DNA sequence(s) to be analyzed; (2) prepare DNA; (3) cut DNA with restriction endonucleases; (4) sort fragments by electrophoresis; (5) visualize the sorted fragments; (6) analyze the results. Types of sequences: (1) nuclear DNAs (nDNAs) – high, moderate, low copy number; (2) organelle DNAs (mtDNA) simple to assay, high copy number, can be separated from nuclear DNAs. Methods of DNA preparation: total DNA versus purified mtDNA Visualization: Direct stain, end-labeling, transfer hybridization. Case Study: The manager of the college food services promised the Chancellor of the college a plate of his favorite chocolate chip cookies for the upcoming meeting with the executive council of the Board of Directors. The chocolate chip cookies, a specialty of Chef Rojo’s, are famous across the college campus. The day before the board meeting, Chef Rojo checked the pantries in the main kitchen and discovered that he only had one bag of the special chocolate chips left he needed for his cookies. He placed all of the ingredients onto the lower shelf of the pantry so that he would be ready to bake the cookies first thing the next morning. Chef Rojo locked the pantry, turned off the kitchen lights and left for the night. When he returned the next morning, he was greeted with a horrific sight. The door to the kitchen had been pried open, and the metal door on the pantry was bent and torn where someone had pried the lock of the pantry door. The ingredients for the cookies were scattered around the kitchen. The bags of sugar and flour were busted on the floor. The open and empty bag of chocolate chips lay on the kitchen counter along with a glass that had lipstick marks on it and a note printed in black marker. The thief had cut his or her hand on the pantry door and left blood stains. Chef Rojo immediately called the campus authorities to report the tragedy. The criminal justice, genetics and chemistry lab instructors were all then called to the crime scene. The investigators interviewed a group of students who had returned home late from an outing and said that they had seen two professors walking near the dining hall at 2:00 am. The professors were Dr. Chandler Bing, in History, and Dr. Arya Stark, in Language Arts. The first suspect was Dr. Chandler Bing because he sat on the executive council as the faculty representative to the board and would have known about the chocolate chip cookies for the meeting. Interestingly, both Dr. Bing and the other professor, Dr. Arya Stark in Language Arts, each had a hand bandaged when they were interviewed. The genetics lab instructor confiscated the black markers that were on each professor’s desk for further analysis. Each professor also gave buccal swab sample for DNA analysis. The following things were set up for the reaction by using the DNA samples from the suspects and the crime scene: Reaction Tube # Crime scene sample Hinc II digest 1 Crime scene sample Xmn I digest 2 Crime scene sample HincII/XmnI digest 3 Suspect 1 sample Hinc II digest 4 Suspect 1 sample XmnI digest 5 Suspect 1 sample Hinc II/XmnI digest 6 Suspect 2 sample Hinc II digest 7 Suspect 2 sample XmnI digest 8 Suspect 2 sample Hinc II/XmnI digest 9 The above is a photograph of the gel obtained. The genetics lab class that the instructor teaches is going to help solve this crime. Lab exercise: RFLP Mapping: Read the instructions about restriction mapping Section VI Restriction mapping. Then construct a restriction map for the data at the top of page 175. Use a fresh sheet of paper to show the drawings. Make sure that you show the fragments cut by each endonuclease, on above the map obtained by the double digest, and one below. Don’t forget to label your map with the fragment sizes. Get this signed by your instructor. Semi-log plotting of RFLP: Complete Section V. Determination of DNA restriction fragment size. Follow the instructions in the section. The semi-logarithmic graph paper will be provided in class and in a downloadable format on Canvas. Plot the control strand and lane 3 and 4. Case Study: SOLVE THIS ONE ALONE. Who is the thief? Lab Report: Using the case study, write a full lab report. In the result, the picture from the gel on this lab manual can be incorporated. Discuss on if you know who is to blame for this crime and what are the evidences. Along with this lab report, attach all the papers you drew the RFLP map on. Also attach the Semi log paper with your graph as part of the report. Do not forget a cover page.

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