Third Assignment Drawing Restriction Maps Predicting Gel Electrophoresis Results Based on Maps Drawing Maps Based on Gel Electrophoresis Results Predicting and Interpreting Results of Southern Analyses Examples: chicken ovalbumin cDNA chicken genomic digest probed with chicken ovalbumin cDNA fragments – initial evidence for the presence of introns Introduction In the first part of this homework assignment, you will use the information you got from NEBCutter to draw a restriction endoncuclease map for the ovalbumin cDNA. Using that map, you will draw expected results for gel electrophoretic analysis of various restriction enzyme digests. Finally, you will draw an alternative map based on those same gel results. The second part of the assignment will place more focus on the ovalbumin gene, as opposed to the cDNA. The chicken ovalbumin gene was one of the very first genes in a higher eukaryote in which intervening sequences were found. Workers in the late 1970s and early 1980s used results from Southern blotting to formulate the hypothesis that the chicken ovalbumin gene must include intervening sequences. They designed additional Southern blotting experiments to further test the hypothesis. Later, when both the cDNA and the genomic DNA had been sequenced, the investigators were able to confirm their results directly. These exercises include the Southern analyses the ovalbumin investigators might have done. Learning Objectives: Concept Knowledge *You will be able to draw a restriction map from sequence and cut site data. *Given a restriction map and/or cut site data, you will be able to draw the expected gel electrophoresis results. *Given electrophoresis results, you will be able to draw possible maps. *Given a genomic DNA Southern blot of a predicted genomic digest, you be able to show where on the blot you would expect to see signal with a full-length cDNA probe. *Given a genomic DNA Southern blot of a predicted genomic digest, you will be able to use selected probes from within a cDNA to help make a more accurate genomic restriction map or to place the coding regions on a genomic map. GUIDED ACTIVITY I Write your answers on a second sheet of paper or document. Remember to make a copy. 1. Based on your NEBCutter results for the ovalbumin cDNA cut positions, draw a linear restriction endonuclease map to scale for the chicken ovalbumin mRNA (cDNA). Use a scale of 1 cm = 500 bp. For the chicken ovalbumin mRNA, the line should be: 1873/500 = ________ cm long. Label the 5’ end of the map “1” for the beginning nucleotide number and label the 3’ end with the appropriate ending nucleotide number. On the map, use your ruler to position the cut sites for each of the following enzymes, as appropriate according to your printouts. Bam HI, EcoRI, HhaI, Hind III, PstI, XbaI, NotI Go on to the next page. 2. a. Imagine that you are going to perform a series of restriction endonuclease digests on the ovalbumin cDNA and run the products of each digest in a lane of an agarose gel. Refer to the key below. For each digest (lane), give the lengths of the fragment(s) you would expect after the indicated digest. Show any math you use to determine fragment lengths. Lane 1: EcoRI was used on the fragment Lane 2: Xba I was used to digest the fragment Lane 3: Pst I was used to digest the fragment Lane 4: both Xba I and Pst I were used to digest the fragment in a single reaction Lanes 5 and 6: blank (no answer required) Lane 7: Commercially prepared size markers (no answer required) b. In the diagram below, draw the gel results that you would predict if you were to subject your digestion products to electrophoresis. The numbers on the right designated the length of markers in base pairs. The markers to 1400 differ by 200 bp, those above 1400 differ by 100 bp. Number the lanes 1—7, starting with “1”, as shown. (You will need to make a copy of this page when you have completed the assignment.) 1 2000 1700 1400 800 400 3. This question is designed to illustrate how single digests can lead to more than one possible map and how double digests, or knowledge of sequence information, can help you distinguish between two possible maps. a. Refer to Lanes 1-3 of the diagram you drew in 2b . In addition to the cDNA RE map you drew based on your NebCutter results, there is a second map that would also be consistent with the gel results in Lanes 1-3. Draw this second map, being sure to ignore both Lane 4 and your sequence information. (Note: this map should not be a mirror image of the first map you drew.) b. In lane 5 of the Diagram above, draw the gel results as they would look if the alternative map you drew for 3a were correct and you had performed a digest using both XbaI and Pst I in a single reaction. (Your answer should look different from Lane 4!) GUIDED ACTIVITY II: Using the computer to map and locate the exons in the chicken ovalbumin gene. II.1. On a separate sheet of paper, draw a linear restriction endonuclease map to scale, using your results from NEBcutter for the chicken ovalbumin gene (not the cDNA). Use a scale of 1 cm = 500 bp. a. For the chicken ovalbumin mRNA, the line was: 1873/500 = ________ cm long. For the gene, the line will be: _____/ 500 = ________cm long. b. Label the 5’ end of the map “1” for the beginning nucleotide number and label the 3’ end with the appropriate ending nucleotide number. c. On the map, use your ruler to position the cut sites for each of the following enzymes, as appropriate according to your printouts. Bam HI, EcoRI, HhaI, Hind III, PstI, XbaI, NotI II.2. d. You should have five Eco RI sites on the genomic map, resulting in six fragments. Number those six fragments 1-6, from left to right. Remember to number the fragments, not the cut sites. a. State your hypothesis as to why there are no EcoRI sites in the cDNA sequence even though there are several EcoRI sites in the gene sequence. State your hypothesis as to why there are no Hind III sites in the cDNA sequence. b. Hint: Annotations can help you determine whether or not your hypothesis has been found to be correct. Find information for the ovalbumin gene J00895.1 again by searching in the nucleotide database at NCBI. Instead of going to the FASTA page, go to the GRAPHICS page. Also, on the GenBank page, scroll down to find FEATURES, and under FEATURES, find mRNA. Look at the “join” information. GUIDED ACTIVITY III: Using Southern analysis to demonstrate the existence of introns and to map genomic restriction fragments. You will not be able to answer this correctly if you have not understood and completed #I.1 and #I.2 above. You are going to reconstruct results that investigators in the late 1970s got from gel and blot hybridization analysis. They did not yet have sequence! III.1. In Lane 1 of the diagram below, sketch the positions in the gel at which you would expect EcoRI fragments 1-6 to be located if you were to digest complete chicken genomic DNA. (Remember that if you were actually to run a gel of digested chicken genomic DNA, it would appear as a continuous smear because there would be so many other fragments in addition to the ones containing the sequence of the ovalbumin gene.) Remember that if you were to actually digest chicken genomic DNA, you would not know the exact positions of Fragments 1 and 6 based on the sequence information you have, because you do not know the location of the next EcoRI site to either side of the mapped region. Put each of these two fragments in any logical place. Reproduce the diagram you made in Lane 1 in Lanes 2-4. At this point, Lanes 1-4 should look identical. In each lane, as specified below, circle the fragments that would be positive after hybridization with the cDNA probe mentioned. Use the comparison of your cDNA and genomic restriction maps, and the annotations you studied in Part II, to help you. Lane 1 – the full-length cDNA Lane 2 – the cDNA fragment 5’ of the Pst I site (think of the slide on subcloning) Lane 3 – the cDNA fragment located between the PstI and Xba I sites Lane 4 – the cDNA fragment located 3’ of the Xba I site. 1 1 2 3 4 10,000 bp 7,0001 4,000 1,000 250 Analyses of Southern Data (the way the existence of introns in chicken genomic DNA were first determined) Imagine that you are one of the early investigators of the ovalbumin cDNA and gene. You have restriction enzyme and gel electrophoretic analysis of ovalbumin cDNA, and you have Southern blotting data of chicken genomic DNA probed with the ovalbumin cDNA and its fragments, but you have no sequence yet. In other words, you have only the experimental information from the gel electrophoresis of the cDNA and the Southern blotting and probing of the genomic DNA as you diagrammed it in this assignment. III.2. Explain why the data shown in Lane 1 of the Southern blot and the data from restriction enzyme digests and gel electrophoretic analysis of the ovalbumin cDNA would lead you to make the hypothesis that the ovalbumin gene must contain intervening sequences. III.3. Explain how the results in Lanes 2-4 of the Southern blot further support the hypothesis. III.4. Explain in words and with a diagram how your cDNA RE map and your Southern results would together allow you to partially determine the order of the genomic ovalbumin restriction digest fragments in the gene without knowing sequence. Note that the answer to this question requires absolutely no knowledge of sequence!