There are 5 questions that need to be answered and one graph. I have uploaded the questions, and a step by step guide to analyze the mtDna.
4 Total = 4361 bp BstN I (130 bp) BstN I (1059 bp) BstN I (1442 bp) BstN I (2500 bp) BstN I (2621, 2634 bp) …CCAGG… …GGTCC…BstN I cuts: BIO 202 Lab#1: DNA Electrophoresis Size Marker: - piece of plasmid DNA (pBR322) - cut with restriction enzyme, BstN I 1857 bp 1058 bp 929 bp 383 bp 121 bp pBR322 DNA Fragments on Agarose Gel: e.g. BIO 202 DNA gel 5 BIOLOGY 202 LAB #1 Human Mitochondrial DNA PCR: PROBLEM SET Name__________________ HAND IN YOUR ANSWERS FOR MARKING BY THE INSTRUCTOR. PLEASE INCLUDE A PICTURE OF YOUR GEL AND ONE GRAPH. (Note: Complete answers will be posted later on the BIO202 Lab Home Page) 1. Observe the photograph of the stained gel containing your sample and those from other students. Orient the photograph with the sample wells at the top. Include a picture of the gel with the marker bands properly labeled and the band for your groups mtDNA labeled. Indicate where the Anode and Cathode are. Make sure to have a proper title and figure legend. a. Scan across the photograph to get an impression of what you see in each lane. You should notice that virtually all student lanes contain one (or more) prominent bands. Some groups may show no bands. b. Now locate the lane containing the DNA markers (pBR322 cut with BstNI). Working from the well, locate the distance traveled by each of the bands corresponding to each restriction fragment: 1857 bp, 1058 bp, 929 bp, 383 bp, and 121 bp (the last band may be faint or not present). - Using the DNA size marker lane make a graph with Log Size (base pairs) on the Y axis vs. distance traveled on the X axis to determine the size of the unknown band or bands in your groups sample (please make sure to have a proper title and figure legend and label the graph properly). Compare the size of your DNA band with other groups DNA bands (label the size on your gel). Briefly explain why the PCR of mtDNA tends to produce a single band of a particular size. ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ 2. For the next questions you will need to analyze the DNA sequence of your groups mtDNA fragment: A) Point your web browser to www.bioservers.org/bioserver/index1.html . Log into the Sequence Server either as a guest (click on enter), or using a registered account (you can register quickly online, which will allow you to save your work for later). FIRST EXERCISE: (NOTE: you can do this exercise any time after the lab is complete) a) Click Manage Groups to open a new window in which you can view mitochondrial control region sequences currently on the Sequence Server database. In the pop-up window SELECT the Sequence sources (e.g. select Public). In the pop-up window SELECT the Appropriate Time Period (e.g. select January to June, 2014). Now scroll down the list of public groups until you reach the group named UFV (12/21/2012). CHECK the box on the left beside the UFV name and CLICK the OK box at the bottom of the pop-up window. CLOSE the pop-up window. You should now see a box with family1 under the Title: UFV, 2012-12-21. CHECK the box on the left. In the box below entitled None, SELECT family2. After you do this another box with None in it will appear. Select family3 from this box. In the next None box select family4. Now you should have FOUR boxes checked with family # 1-4 in them. These are mitochondrial DNA (mtDNA) sequences from a family of a husband and wife and TWO daughters. b) CLICK on the OPEN box beside one of the sequences to view the sequence in another pop-up window. You will notice that the sequence contains A, C, T, G’s as well as N’s. The N’s are nucleotides which could not be determined by the sequencing procedure. These tend to be found mainly at the ends of the sequences where the sequencing reaction doesn’t work as well. (NOTE: If these are found in abundance throughout the sequence then the DNA was probably contaminated or at too low a concentration.) CLICK Done when you are through. c) You can compare these four sequences to find the similarities and differences. CLICK on the COMPARE box at the upper left part of the page (align: Clustal W) with all four sequences checked. A new pop-up window opens which will show all four sequences aligned together to show the similarities and differences. (If another pop-up window warns you about errors, don’t worry about it. Just close the warning window.) Scroll down to look at the four sequences. You will note that there are a lot of differences at the beginning and end of the sequence. To remove these parts check the trimmed circle and CLICK on Redraw. (Ignore any warning pop-ups). http://www.bioservers.org/bioserver/index1.html 6 d) Which sequence is from the FATHER? How do you know? How many differences (yellow) are there between the father and mother? (NOTE: don’t include parts of the sequence where there are N’s present, or where the four sequences are not ALL present.) ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ ______________________________________________________________________________________________________ SECOND EXERCISE: (NOTE: This can ONLY be done once YOUR mtDNA has been sequenced which can take several weeks after the samples are sent off.) a) Click Manage Groups to open a new window in which you can view mitochondrial control region sequences currently on the Sequence Server database. In the pop-up window SELECT the Sequence sources (e.g. select Public). In the pop-up window SELECT the Appropriate Time Period (e.g. select January to June, 2016). Now scroll down the list of public groups until you reach the group named UFV BIO202 mtDNA data 2016. CHECK the box on the left beside the UFV name and CLICK the OK box at the bottom of the pop-up window. CLOSE the pop-up window. Now, SELECT your mtDNA sequence (use your assigned number and lab section or instructor) and check the box on the left. If your mtDNA is not present, then use ANY sequence to complete your assignment. b) CLICK on the Manage Groups button and then SELECT the Modern human mtDNA sequences in the pop-up window and select all the different races mtDNA. Then click OK. The window will close, and the selected sequences will now appear in a list in the Sequence Server workspace. c) CLICK on the Manage Groups button and then SELECT the Non-human mtDNA sequences in the pop-up window and select primate mtDNA. Then click OK. The window will close, and the selected sequences will now appear in a list in the Sequence Server workspace. d) CLICK on the Manage Groups button and then SELECT the Neanderthal mtDNA sequences in the pop-up window and select at least 5 different types of Neanderthal mtDNA then click OK. The window will close, and the selected sequences will now appear in a list in the Sequence Server workspace. e) In the Sequence Server workspace, visually gauge the quality of your group’s mtDNA in the following way: - Use the pop-down menu displaying the sample names to select a student or teacher sequence. Then, click the adjacent View to open a new window showing the nucleotide sequences of the selected entry. Every sequence will begin with nucleotides (A,T,C,G) interspersed with N’s. indicating that the nucleotides could not be determined at these positions. In "good" sequences, where experimental conditions were near optimal, the beginning N’s will end abruptly. The remaining sequence will have very few, if any, internal N’s. Then, at the end of the "read," the sequence will abruptly change over to an uninterrupted string of N’s. In non-optimal cases, a large number of N’s will be interspersed throughout the sequence. When possible, use good sequences (those without internal N’s) in your subsequent comparisons. If your sequence has too many N’s throughout then you may use another groups mtDNA. f) Now, compare your sequence with another group’s mtDNA sequence using the ClustalW algorithm available in the Sequence Server workspace. CLICK in the check boxes to the left of the two (or more) sequences you want to compare. Then CLICK on Compare to send your sequence to the ClustalW alignment tool and align the checked sequences. Your results will be returned in a new window. Once the sequences are aligned there will be significant similarity between the sequences except for the beginning and the end of the sequences. Redraw the alignment after checking the Trimmed function and the ends of the sequences will be cut off giving a more appropriate comparison. g) Count the number of differences (in the good parts of the sequence; without a lot of N’s) - A yellow box indicates positions with a nucleotide difference. A gray box indicates positions with an N, where a nucleotide could not be determined. Read into the sequence until you get past the initial stretch of interspersed N’s. Then begin counting the number of yellow highlighted nucleotide differences. Do not count any internal N’s. Also count dashes (-), which indicate a deletion, a nucleotide that is absent at that position in the sequence. Record the number of differences (see figure BELOW!). 7 h) Repeat the above steps f) and g) and compare your mtDNA to other students and other modern humans (make at least 7 other comparisons) to get an average number of mutational differences in the control region mtDNA between different modern humans. (SHOW YOUR CALCULATIONS BELOW!) i) Since we know that modern human races most likely diverged about 150,000 years ago using a number of different types of analysis (including mtDNA analysis) we can determine a mtDNA mutational rate clock by dividing the 150,000 years by the average number of mutations in the human mtDNA. Determine the mutation rate (number of years for one mutation): __________________years/mutation ______________________________________________________________________________________________________ _______________________________________________________________ j) Now compare your groups mtDNA to the mtDNA from several (at least 5) Neanderthal’s. Count the number of differences in the mtDNA sequences and average them. Approximately how long ago did we diverge from Neanderthal’s using the mitochondrial mutation clock?