Personal Exam Code: 1. 16p

Transcription

1 Exam, Methods in Molecular Biology, BIOR47, Please hand in answers to each question on its paper. Use additional papers if needed. Mark all papers with your code. Your answers should be logical and concise, do not write long essays! Give reasons for your answers and include all the relevant information! Good luck/ Allan 1. 16p Briefly explain the following terms/techniques: a. SDS-PAGE b. BAC c. Cosmid d. Northern Blotting e. Gateway cloning f. Phage display g. Metabolomic analysis h. SNP analysis

2 2. 8p This is a question about RNA. The image denotes agarose gel electrophoresis patterns (RNA was loaded at the top) of isolated Qiagen-purified RNA from 5 different organisms. A) One sample is from a prokaryote. Which? Motivate. B) Denote in the figure where the major non-coding RNAs and mrna should reside in the left-most lane. C) If there would be contaminating DNA, where (approximately) would you see it in the gel? Denote in the figure. D) If the RNA in the leftmost lane would be partially degraded in the tube, what changes should you expect when analysing again? E) Describe two different types of RNases, and what reactions they carry out.

3 3. 7 p First, define amplification efficiency. Then, describe for each of the following problems (a-c), how it will affect (1) the specificity and (2) the efficiency for a PCR reaction to produce a single product. Motivate in each case why the specificity and/or efficiency should be effected. a. One primer ends with the sequence GACCCG-3. b. One primer in the pair has a Tm that is 8 C higher than the Tm of the other primer. c. One primer ends with the sequence AGTCGACT-3

4 4. 6 p These questions concerns Western (immunoblotting) analysisa) Describe the principle behind Western blot. b) Describe the use of a primary and secondary antibody in a Western blot. c) When do you not need to use secondary antibodies in a Western blot? d) You are interested in Mitogen activated protein kinases (MAPK). You have two type of antibodies; mouse anti MAPK antibody and a rabbit anti phospho MAPK antibody. How would you set up a multiplex western blot assay with these antibodies.

5 5. Micro array analysis 3p I: It is necessary to normalize microarray data because: A. Gene expression values are not normally distributed. B. Some experiments use cdna labeled with fluorescence while others employ cdna labeled with radioactivity. C. The efficiency of dye incorporation (or radioactivity incorporation) may vary for different samples. D. Housekeeping genes (such as actin) may be expressed variably between samples. II: Microarray data analysis can be performed with scatter plots. The information you get from a scatter plot includes all of the following EXCEPT: A. You can tell whether a gene is expressed at a relatively high level or a low level. B. You can tell whether a gene has been up regulated or down regulated. C. You can tell whether a gene forms a cluster with other genes on the microarray. D. You can tell whether a gene is among the 5% most differentially regulated genes in that experiment. III: Log ratios of gene expression values are often used rather than raw ratios because: A. Twofold up regulation or two fold down regulation log ratios each have the same absolute value. B. Twofold up regulation or two fold down regulation log ratios each have the same relative value. C. The scale of log ratios is compressed relative to the scale of raw ratios. D. A plot of log ratios compresses the expression values.

6 6. 6p These questions concern DNA sequencing technologies a) Describe the principle behind the chain termination (Sanger) method of DNA sequencing. b) The chain termination method represents the first generation sequencing methods. Give one example of a second generation method and briefly explain how it works. c) The second generation sequencing methods allow incredibly rapid generation of large amounts of sequence data. Why is Sanger sequencing still widely used for DNA sequencing?

7 7. 6 p a) In the manuals for the rotors of the preparative ultracentrifuge so called clearing factors (k) are given. Explain what this factor could be used for? Give the equation (mathematical relationship) that will be employed and what are the units of all factors of the equation? b) Separation of particles will be achieved according to the methods of: differential centrifugation, rate-zonal sedimentation and equilibrium density centrifugation. In which of these techniques is the k factor most often applicable?

8 8. 6 p You can use the green fluorescent protein (GFP) as a reporter to address a large number of questions. Below are listed three different aspects of a gene (e.g. yfg) or its gene product (Yfg) in a specific microorganism that can investigated using fusions to GFP. For each of the cases, describe briefly how you can use GFP to answer the question! What kind of fusion has to be created? Which part of the gene has to be fused to the gene for GFP? How do you detect the signals and what can they tell you about the gene yfg? (a) When is the gene yfg transcribed? (b) Where in the cell is the gene product Yfg localized? (c) Is the protein Yfg freely diffusible within the cell?

9 9. 6 p Describe on a cellular and molecular level the different steps that takes place when a plant is infected by Agrobacterium tumefaciens in nature (6p).

10 10. 6 p What is meant by complex trait? Give an example of a disease which is considered a complex trait.