1. Using BLAST to Identify Species from Proteins
Adapted from College Board’s “Investigation 3: Comparing DNA Sequences to Understand Evolutionary Relationships with BLAST”

2. What we know so far...
We already know that DNA can be used to identify animal species at a genetic level
According to the Central Dogma of Biology, DNA is translated into RNA and transcribed into proteins
If DNA is not present in a bone specimen, other biological material can be acquired to identify a fossil

3. Today’s Goal Become acquainted with BLAST for protein analysis
Identify the relation of extinct dinosaurs to modern species and where dinosaurs fit in on a family tree
Understand how phylogenetic trees depict evolutionary relationships

4. What you will need to know
What is BLAST, and how is it used?
Why is BLAST important?
What is a phylogenetic tree?
What are orthologs?
How can proteins be used to identify evolutionary relationships?

5. What is BLAST?
B L A S T
Basic
Local
Alignment
Search
Tool

6. Why is it important?
BLAST is a tool connecting databases that contain many millions of partial, full, and potential gene sequences
It can be used to compare sequences from one species to another to identify similarities or differences
Knowing the identity of gene sequences from many species allows scientists to track evolutionary heritage of those species

7. What is a phylogenetic tree?
Diagram that depicts the evolutionary relatedness of species or groups of closely related species
Each endpoint of a branch represents a group of organisms
The closer two branches on a tree are, the more related the groups are
More information about phylogenetic trees is present in the student activity.

8. What are orthologs?
Orthologs are genes in different species that evolved from a common ancestral gene
Basically, they are genes with the same or similar functions in different species
Ex. Human and mouse genes
Gene duplication occurs during DNA replication. When species differentiate, the same genes are duplicated across species. The alpha chain of a gene is the same among frogs, humans, and mice. Therefore, these species are orthologs.

9. How can proteins be used to identify genetic relationships?
Proteins are composed of different combinations of amino acids
The genetic code (DNA/RNA) is used to identify each amino acid to then add on to a growing chain (protein)
There are 20 amino acids and 64 possible combinations of RNA building blocks- some amino acids are repeated!
These repetitions allow scientists to see the differences between proteins
Each amino acid is represented by a single letter “code” so scientists do not have to write out the full name. For example, “Leucine” is “L”, while “Lysine” is “K.” A full listing of each amino acid code can be found in the supplementary material for this activity.

10. Activity: Identify Evolutionarily Related Protein Sequences
You are a member of a scientific team that has discovered 3 well preserved bone fossil specimens
T. Rex, Mastodon, and Hadrosaur
Small amounts of tissue have been removed from the fossil
Unusual in such ancient specimens
Amino acid sequences from protein fragments have been extracted
First time this has ever happened!

11. Activity: Identify Evolutionarily Related Protein Sequences
Use BLAST to compare these amino acid sequences to protein sequences from other species
Then, create a phylogenetic tree to explore where these extinct species might branch off from the evolutionary tree

12. Step 1
Form an initial hypothesis as to where and why you think the extinct specimens belong on the phylogenetic tree

13. Step 2 INSERT IMAGE OF LOCATION OF PROTEIN FILES FOR SMILE
Locate and download the protein fragment files for the fossilized bone specimens

14. Step 3 Upload the sequence into BLAST
Go to BLAST homepage: st.cgi
Click on “Protein BLAST”

15. Step 3
Under “Enter Query Sequence,” paste the first amino acid sequence from “BLAST Protein Fragments”
Do NOT change any of the settings on the rest of the page
Scroll down and hit “BLAST”

16. Step 4 The results page has 2 sections
The first is a graphical display of the matching sequences

17. Step 4
Scroll down to the section titled “Sequences Producing Significant Alignment”
The species in this list are the ones with sequences identical to or most similar to the amino acid sequence of interest
The most similar sequence is listed first
If you click on a result listed, you will get a full report of the species

18. Step 4
This is the results page “Sequences producing significant alignment.” The blue links are the most likely matches for the sequence you placed into BLAST. The first result is the T. rex sequence match, confirming you have placed the T. rex sequence in the search. The next few results are split into 2 categories: predicted and other. Predicted is what the database thinks is the best match, but ignore that as it is hypothetical. Instead, focus on the other results in the top 10 sequences.

19. This is an example results page for the first result on the T
This is an example results page for the first result on the T. rex search page. All the answers are provided under the headings on the left.

20. Now that you have this information...
Follow the PDF instructions for the BLAST search of the fossilized T. rex bone amino acid sequence
If you still have time at the end of class and want to practice and explore more, repeat the activity for the hadrosaur and mastodon fossil specimens