Experiment 2 E2.1 Experiment 2: Genome Variation Part 1 - Finding TE insertions in B73 using bioinformatics The sequence of a transposable element will be used as a BLAST query in the B73 maize genome browser. From the hundreds of hits TE insertions in introns will be chosen using the following criteria:  Only 1 TE insertion in the intron.  Intron less than 1 kb.  EST (Expressed Sequence Tag) support for the exons flanking the intron. The Maize Genome Browser at the Phytozome website will be used to annotate TE insertions. In lab, primers to the flanking exons will be used to amplify the intron region from B73 and many other strains of maize. 1. Open a web browser to Phytozome (www.phytozome.org) and click on the gray square “Click here to search…”. 2. Close the popup gray box and select Zea mays on the tree. 3. In the Choose a tool, click “Expand” next to the BLAST Search option. Experiment 2 E2.2 4. In the box, a) select “Genome” as the target, b) copy and paste a TE query in the Query text box and c) click “Submit”. The queries can be found on ilearn in course materials. c. a. b. 5. In the results window click “Switch Views”. 6. In the new results window, click on a good Blast hit. A browser window will open zoomed in on the blast result. Zoom out to 5 kb by clicking on the “Show n bp” menu to see if the hit is in a gene. 7. Using the information in the browser, does the hit meet the criteria stated above? If so keep the screen and be prepared to discuss the genomic region. Experiment 2 E2.3 Now that intronic TE insertions have been found we need to design a PCR experiment to amplify the region out of genomic DNA samples. We will do a thinkpair-share to design the experiment. Part 2 – PCR Set-up The PCR protocol for this activity is similar to the one used in experiment 1. There will be DNA samples from many different strains of maize and each student will choose three. Design a PCR experiment using the one in experiment 1 as a model. What other reactions should be included? What reagents are needed? Write the protocol in the lab notebook. Notebooks will be checked before class. Part 3 – Gel Electrophoresis Follow the protocol from experiment 1 and pour one gel with two combs per bench. Gels will be stored at 4˚C until the next class. During the next class, load 5ul DNA ladders in the first lane and 12 µl of each sample. Part 4 – DNA Sequence Analysis After gels are run during the next class the DNA sequences of the PCR amplicons will be placed on the website. What questions can be addressed using this sequence? What bioinformatics tools will be needed to address these questions? Learning Goals Students will: 1. Be able to describe three types of genome variation-SNPs, indels and TEs 2. Be able to describe the source of the variation. 3. Be able to design and set up a PCR experiment to amplify across and intron. 4. Be able to identify variation though comparative DNA using BLAST and multiple sequence alignment programs.