Purpose:

We will use the tools of the World Wide Web to search the GenBank DNA sequence database for similarities to any given DNA sequence. For this exercise, you will use the DNA sequence that you derived by "reading" sequencing autoradiograms.

Skills and content areas

• Use of the World Wide Web to access national data bases
• "Reading" of DNA sequencing outputs (gels)

We will use the tools of the World Wide Web to search the GenBank DNA sequence database for similarities to an unknown DNA sequence. You will need an unknown DNA sequence for this exercise. This sequence can be given directly to the students on a diskette (they will copy / paste the sequence into the DNA search form on the Web program) or you could draw a pretend autoradiograph of a DNA sequencing gel (see below) and have the students "read" and enter the base sequence.

SAMPLE DNA SEQUENCE: Underlined portion of this sequence is shown in the above "gel" figure TCCCAGATCACTGTCC TTCTGCCATGGCCCTG TGGATGCGCCTCCTGC CCCTGCTGGCGCTGCT GGCCCTCTGGGGACCT GACCCAGCCGCAGCCT TTGTGAACCAACACCT GTGCGGCTCACACCTG GTGGAAGCTCTCTACC TAGTGTGCGGGGAAC GAGGCTTCTTCTACAC ACCCAAGACCCGCCG GAGGCAGAG         Note: using a larger sheet of paper, you can "show" more of this sequence by extending the figure at the top!

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Performing the sequence search:

We will be accessing the national database of DNA sequences, GenBank, using the World Wide Web and the Netscape search application. For those of you who have not used Netscape, please speak with the instructor for guidance on how to start the program.

1. Access the National Center for Biotechnology Information web site: http://www.ncbi.nlm.nih.gov
2. Choose BLAST sequence similarity searching.
3. Choose Basic BLAST search.
4. Choose the program [blastn=nucleotide; blastp=protein] and the database [nr=nonredundant] for your search. Enter the unknown sequence (i.e. from your autoradiographs) in the form box by either typing directly or by copy / paste from a text document (i.e. MS Word file). This is the "query".
5. Submit query and wait for reply.
6. Observe BLAST search results, listed first by name and score, followed by a visual alignment of the match. **Be sure to scroll down to "see" the actual alignments!**
7. Clicking on the highlighted, high-scoring segment pairs will provide access to a wealth of information on the gene matching your unknown sequence, including the entire gene sequence and reference publications. Go ahead and play with it.

At the end, you should know:

• What did your sequence match in the database? (i.e. what gene, from what species).
• What degree of match did you find? (i.e. % similarity / identity)
• Do you think you really have isolated the sequence for a known gene? Why or why not?
• Who are the authors (discoverers) of the known gene that matches your sequence? Did they publish their work? When?

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