Now that you have the wild type beta-globin sequence, you can use it to find the mutant beta-globin sequence. Because we know that the mutant beta-globin sequence differs from the wild type sequence by a single base, we can use the wild type sequence to search a database for sequences that are extremely similar (or homologous) to it. In order to carry out the homology search, you are going to use a sequence alignment tool called BLAST.

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1. Choose the appropriate version of BLAST. BLAST is composed of a series of programs for searching sequences in databases; make sure to choose the BLASTN program that performs the search with a nucleic acid sequence in a database of nucleic acids.

Go to the session called Sickle Cell Anemia

Select the line GBPRI:29436 Human messenger RNA for beta-globin from the list of sequences available within this session

Scroll down the window until you see the line BLASTN - Compare a NS to a NS DB

Highlight that line

Click the Run button

2. Setting up the BLASTN search parameters.

In the Query strands to search, do not change anything

In the window with databases, you can select one or more databases; highlight ALL the lines for the GenBank Primate Sequences

Leave the rest of the parameter boxes unchanged.

Scroll down to the bottom of the screen and click Submit button in order to run the search

3. Examining the results of the BLAST search. This screen may take some time to show up. It could be short or long depending on the number of hits that the search process found.

Regardless of the number of hits; the BLAST results screen will be structured in this way:

Top of the screen will report the values you provided for the search like the target sequence and the database(s) searched.

The next section of the screen reports the number of hits found during the search

The next section of the screen reports the reference of the BLAST algorithm used

The next section of the screen has the set of commands that you can use on the hits;

these commands are: Show Record(s), Show Sequence(s) Import Sequence(s), Return, Help, Report Bugs

The next section of the screen is the summary report; it is given in table form.

The columns of the table are: Select, Database, ID, Name, Score, Evalue

The rows of the table are the hits found in the search

The next section of the screen is the detailed report; for each hit, there is pertinent information regarding the matching of it with the target sequence

The last section of the screen provides information on statistics used during the search

Notice that the first result listed is in fact the wild type sequence that you searched with. If you look over to the "Score (bits)"column, you will see that the first few sequences have very high Score (bits) values. A Score (bits) value above 400 means that the sequence has high homology with the sequence you are comparing it to. However, you can be more certain of the extent of the homology between two sequences by looking at the "E Value". This is the number to the right of to the "Score (bits)" number. Essentially, the smaller the E-value, the more homologous or similar the sequence is to the original sequence BLASTED. An E-value of zero indicates that no matches would be expected by chance - this would represent a perfect or near perfect match.

4. Selecting the mutant sequence. Now it is time to decide which sequence is the mutant beta-globin sequence. Since you know that the mutation is a small one (a so-called point mutation) this means that the wild type and mutant beta-globin sequences should be nearly identical (have very high homology). Using this information, a good way to find the mutant sequence is to highlight all of the sequences with high "Score (bits)" values and "E Values"of 0.0, excluding the first sequence because this is the wild type beta globin sequence. Then click on the "Show Record(s)" button to get more information about the sequences that you have highlighted.

Scroll down the "Records"window until you find the beta-globin mutant sequence. Remember that the mutation that you are looking for causes sickle cell anemia so the world "sickle"may be in the description of the sequence.

Scroll down the screen until you find [in the summary report section] the sequence

GBPRI: 183944 Human sickle beta-hemoglobin mRNA M25113

Select it by putting a check mark in the first column of that line/row

Scroll up the screen to the commands section and click Show Record

5. Importing the mutant sequence. Remember that the Biology Workbench does not copy data onto your session in an automated way; you have to choose which data to store in your session.

Once the screen shows the record corresponding to the mutant sequences

Scroll down the screen to examine the details being displayed

Go to the bottom of the screen and click Import Sequence to copy the sequence to your session

Another screen will appear; click Return to go back to the Sickle Cell Anemia session page

Make sure there are now two sequences in your data list: one for the wild type and one for the mutant sequence.