Comparing Your Sequence to a Nucleotide Sequence Database
This database will compare your nucleotide sequence to a database of nucleotide sequences and identify potential matches. If you had an unknown sequence this is one step you might use to identify your gene, however, since you already know the identity of your gene, this search is useful for finding nucleotide sequences that are homolgous to your sequence. You may find that your gene belongs to an entire gene family or that it is conserved across many different species.
Beginning Your Search:
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Click on the link below to obtain the beta-globin sequence. When the screen opens, first select the "Select All" and then "Copy" commands under the Edit menu. Then use your browsers back button to return to the BlastN search page. Use the "Paste" option under the edit menu to paste the beta-globin sequences into the search box.
- Do not enter anything in the boxes for "Set subsequence"
- Make sure that "Choose database" is set for "nr" or non-redundant. This option will exclude matches that consist of repetitive DNA sequences from your search results.
- Scroll further down the page to the "Format" Options box and change the Alignment Number form 50 to 100.
- Click on the "Blast!" button to begin your search.
- When the next screen comes up, click on the "Format!" button. Considering the size of the database, your search will not be completed immediately. The page will automatically refresh itself until the results are available.
Interpreting Your Search Results:
- The top of your results page will display references related to your gene of interest. Scrolling further down the page will first display a Color Key of the search results based on sequence alignment. These colored bars are a graphic representation of your search results and each bar represents a result that matches your sequence. Longer bars in red at the top of the chart are search results with a high sequence homology to your gene. Pink, green, blue, and black bars follow below representing search results with decreasing amounts of homology. The position and color of each bar is relative to the linear map of your search query and allows you to instantly see how the search results line up against your sequence.
- Below the chart is a list of your search results with assigned number values (Bit score and E value) indicating the degree of homology between your gene and the search result. The bit score is the raw score given to each search result based on homology and normalized to a standard set of units by taking into account the statistical properties of the scoring system. The E value is an "expectation value." The database first determines how likely it is that the match between your search query and the search result occured by chance. The lower the E value the less likely that the match occurred by random chance and the more significant the alignment.
- Take a look at your search results:
1. Examine your search results for patterns. Rank the following as to which displays greater similarities to beta-globin. (HINT: search results are given in order of decreasing similarity from the top.)
Allelic varients: Wild-type beta-globin, Sickle Cell Beta-globin, Beta-thalessemia
Family Members: Beta-globin and Delta globin
Primate Homology: Human beta-globin, Gorilla beta-globin, Chimpanzee beta-globin (Pan troglodytes), Orangutan beta-globin (Pongo pygmaeus)
Non-primate Homology: Human beta-globin, Rabbit beta-globin, Rat beta-globin
2. Average the percent homology for each group and record below (be careful not to include human beta-globin in your average!). Which group displays the greatest average homology to human beta-globin?
3. Now rank the groups based on their averages. What does this rank tell you about how the beta-globin gene may have evolved across species? (i.e. Is human beta-globin more similar to primate or non-primate genes?
4. Compare the sequence homologies among beta-globin family members. Does this information support your predictions made in Entrez Nucleotide Question #6? How might you explain the fact that epsilon and gamma globin do not appear as homologous matches to beta-globin?
When you are finished, return to the Beta-thalessemia Menu Page to move on to the next section of the laboratory.
Beta-thalessemia Menu Page
