BioinformaticsProtocolanddatasheet_CV.docx - Bioinformatics...

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Bioinformatics Lab Objectives 1. Appreciate the number and diversity of genomes and sequences stored in the NCBI databases. 2. Use BLASTn to identify an organism through sequence comparison. 3. Construct a phylogenetic tree using nucleotide sequences. 4. Use MEGA to align protein sequences and develop a gene phylogeny. Safety 1. Closed-toe shoes must be worn Lab Activity 2: Sequence Searching 1. Point your internet browser to the BLAST URL provided by your TA/LI and set a bookmark. If you get lost along the way, you can always come back to the home page and work your way back in. 2. Ask your TA to give you your sequence number. Use this to obtain your individual sequence for this exercise from eCampus. Copy the text file onto your computer. The sequence you receive will be unique to you, so while the procedures your lab group will be conducting will be the same, the results of your work will be quite different. This is exciting! 3. Click on “Nucleotide BLAST” You should see a page like this: 4. Open your sequence file, and copy and paste all of the text into the “ Enter Query Sequence ” text box on the BLAST page. 1
5. Under the “Database” portion of the page, pull down the menu and select “ nt (nucleotide) ”. Make sure the “show results in a new window” is selected. 6. Click the “BLAST” button on the lower left side to start the search. NCBI will return a page telling you the search is in progress. The search may take a few minutes depending on server load—Do not keep submitting or refreshing the page. 7. When the results page appears, scroll down to the “ Descriptions ” section. The first result likely represents your unknown sequence. The top hit is most likely the sequence from your organism. Record the genus and species in the data sheet below. Click on the description link for the first entry. 8. Observe the “ Alignment ” section for your sequence. Record the score, expect value, and identity score in the Lab Activity 2 Datasheet below. As you observe the alignment, note that dashes between the query (your unknown) and the subject (sbjct) indicate identity—a perfect match between the two sequences. Are there any regions of your sequence that don’t have dashes? 9. Click on the “ Sequence ID ” at the top of the entry. Answer the questions for this section on your lab data sheet. 10.Run your BLASTn search again (start at #4) using the sequence given to you, but this time, only use the first line of your DNA sequence as your query . Did your results change? Answer the questions on your data sheet. BEFORE STARTING! Visualizing DNA using MEGA software. MEGA is a program preferred by many phylogeneticists because of its simple and elegant interface for visualizing and aligning DNA sequences.

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