BLAST primerPrepared by Brian Rybarczyk, PhD

Department of Biology

University of North Carolina – Chapel Hill

September 2003

BLAST (Basic Local Alignment Search Tool) is a common tool used in bioinformatics and is free to use on the internet. This tool was created by the National Center for Biotechnology Information (NCBI). Researchers and scientists from around the world use this tool on a daily basis. Although BLAST and the related search engines provided by NCBI are user friendly, this primer will help work through the steps needed to run a basic alignment search. You can find our more about BLAST and related search engines by clicking the links on the NCBI website. For more advanced search options and a more detailed description, click on the BLAST tutorial on the main NCBI site.

STEP 1: Go to www.ncbi.nlm.nih.gov using your web-browser. Click on BLAST.

You must now decide which search engine will serve your needs. If your sequence is DNA, use the Nucleotide BLAST. If your sequence is a protein sequence, use Protein BLAST. Other, more advanced search options are also available on the site.

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STEP 2: Enter your query sequence into the Search box. You can copy &paste your sequence from another file (Word, Sequencer, MacVector). Then click the BLAST! Button.

STEP 3: Once your data is entered and sent to BLAST, in order to retrieve your results, you need to format your data. Click on the Format! Button to do this. Depending on how many users are sending information at the same time, this process could take a few minutes, be patient!

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STEP 4: Once your results appear, a colored chart showing the sequencematches appears. Each line represents a sequence that matched with your sequence. Red is a sequence that has high similarity to the query sequence and black is a sequence that has little similarity to the query sequence. Mouse over each line, the name of the sequence will appear in the description box above the color chart.

linked to more information about each record

clone name assigned by researcher

description

each score is linked to a diagram that shows the query sequence aligned with the matching sequence from the database

Accession number:Unique identifier used to retrieve this sequence from the database

links to other databases

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Clicking on one of the highlighted (blue) search results will bring up an annotation of that particular sequence match from your search. An annotation is a description of a sequence. The information includes the full name describing the sequence, the unique GenBank accession number to identify the sequence, a reference that originally cited the sequence, the organism the sequence is derived from among other descriptors of the sequence.

Scrolling down the annotation, you will find the associated protein sequence as well as the DNA sequence for this entry.

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Introduction to ProteomicsInvestigation into protein structurePrepared by Brian Rybarczyk, PhD

Department of Biology

University of North Carolina – Chapel Hill

September 2003

The NCBI website also links to other search engines that contain protein structure information. The main access for searching available protein structures is through ENTREZ. ENTREZ searches the Protein Data Bank (PDB) for structures that match a query. The information in PDB is derived from crystal structures of proteins. Once a protein structure matches your query, a PDB file (designated by a four letter/digit identifier) is downloaded and can be viewed on your computer. The 3-D view of the protein aids in understanding how the structure and the function of a protein are linked.

STEP 1: On the main NCBI website, click on “Structure” at the top on the dark blue menu bar. Type in the name of a protein you would like to view, you can include the species of the protein you are interested in. Click “Go” and this will retrieve a list of matching and related proteins to your query. Clicking on a blue highlighted 4 digit identifier will bring up another window for that particular protein structure.

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STEP 2: Clicking on one of the PDB identifiers will lead you to a description page of the entry. Click on the PDB Id identifier link and this will lead you to a page where you can choose what else you can do with the file and how to view it.

click here to view the structure

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STEP 3: The next window gives options to view the query structure. Protein Explorer is recommended. It only requires a plug-in called Chime which you can download onto your computer (click “Chime” to install plug-in onto your computer).

STEP 4: click on Protein Explorer

STEP 5: Click on “Start Protein Explorer 2” button on the next window

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STEP 6: The resulting 3-D picture is an image of the query protein. By right clicking using your mouse, you can change the different parameters to view the structure. You can change to ball & stick view, ribbon view, change colors of the structure, highlight certain atoms, control rotation, etc. Also provided is detailed information such as the number of atoms, bonds, helices, etc. Here is your chance to be up-close and personal with your protein of interest!

Thought Questions:Describe the structure of your protein (globular, linear, α-helices, β-sheets, number of subunits, etc).

What does the structure of your protein tell you about its function?

What other proteins are homologous to your protein?

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