EL's+Bioinformatics+procedure

1.  Learn the information on NPC2 disease and NPC2 protein. 2.  Learn how to read protein sequence how the DNA/RNA sequence is formed. 3.  Use internet databases to retrieve protein sequences—ExPASy and NCBI (1) Using ExPASy website a.  Enter address [|www.expasy.org/sprot/], the Swiss-Prot database home page. b.  To get the sequence of human NPC2 protein, type human NPC2 protein in the Search window, and the click the Search button. c.  Select NPC2_HUMAN (P61916) link. d.  At the bottom of NPC2_HUMAN (P61916) page, the sequence section provides you with the actual amino-acid sequence of protein. e.  Click P61916 in FASTA format link to see input sequence data. : The FASTA format of sequence is the default input format for much sequence analysis software, including BLAST and clustalW. f. To get the sequence of cow NPC2 protein, repeat the steps from b to e. Type bovine NPC2 protein. The entry name for cow NPC2 protein is NPC2_BOVIN. g. Save the sequence data. (2) Using NCBI website a. Enter address [|www.ncbi.nlm.nih.gov], National Center for Biotechnology Information webpage. b. In the Search drop-down menu, change All Databases to Protein. c. Enter human NPC2 protein in the Search window. d. Click Sequence tap. e. Save the sequence data. 4. Once you get the sequence data, use BLAST to get further information. a. Enter address [|www.ncbi.nlm.nih.gov/blast/]. b. In the basic BLAST section, click __protein blast__ link. c. In the Enter Query Sequence window section, put FASTA format of NPC2 protein. d. In the database drop-down menu of Choose Search Set section, change Non-redundant protein sequence (nr) to Swissprot protein sequence (swissprot). e. In the Algorithm of Program Selection section, select blastp (protein-protein BLAST). f. Click BLAST button. g. Get more information on NPC2 proteins using a graphic display, a parameter and an alignment. 5. To compare the sequences, use Dotlet program which draws dot-plot. a. Copy each FASTA format sequence, which you found at previous step, of human NPC2 protein and cow NPC2 protein by using ctrl-c. b. Open a new web browser window and enter address [|www.isrec.isb-sib.ch/java/dotlet/Dotlet.html]. c. Click input button. d. Put name of the sequence and paste human NPC2 protein FASTA format sequence you have. e. Click the OK button in the pop-up window. f. Input cow NPC2 protein FASTA format sequence by following step c-e. g. Back in to original browser window, choose human NPC2 protein (P61916) and cow NPC2 protein (P 79345). h. If the default output of Dotlet is difficult to interpret, adjust settings by changing the zoom factor, the window size and the threshold. i. Interpret the regions of sequence similarity of two different NPC2 proteins using the dot-plot graph. k. Compare the results to those of Professor Xiaowen Hou. 6. To compare various NPC2 proteins at a time, build a multiple sequence alignment by using clustalW—gene tree making program. a. In order to get various sequence data, enter [|www.expasy.ch/tools/blast]. b. Enter the Sequence Accession Number P61916 and select blastp for protein sequence. c. Click run BLAST button. d. Scroll down the page to select approximately 8 to 10 NPC2 proteins. e. In the Send send selected sequences scroll-down menu, choose Retrieve sequences (FASTA format). f. Copy all FASTA sequence information on the current webpage. g. Open a new web browser page and enter address [|www.ebi.ac.uk/clustalw]. h. Paste the sequences you collected in the step f. i. Use the Output Format pull-down menu to set the selection of your choice. k. Choose Input from the Output Order pull-down menu. l. Do not select a tree type. m. Click the Run button. n. Save your results and explain the relationship of various NPC2 proteins by analyzing the tree—especially Phylogram Tree.