Reference no: EM132311177

Cells and Genes - Bioinformatics Assignment

Learning Outcomes (Aims):

1. To gain experience in using the National Centre for Biotechnology Information website, which gives access to numerous nucleic acid and protein databases, and much more.

2. To identify the genes or proteins that an unknown DNA or amino acid sequences are part of.

3. To appreciate the differences between nucleotide sequence (e.g., of a gene) and amino acid sequence (e.g., of a protein), and discern properties of such sequences.

4. To manage your time effectively and work within tight time frames.

Bioinformatics Assignment - Gene Identification: An Introduction to Bioinformatics and Databases

Introduction/Background: You are a member of a laboratory that has is involved in the commercial sequencing of genomes and proteomes from various organisms. Some unknown samples and data files have been unearthed. Your supervisor has asked you determine which organism/s the data comes from and other information about the sequences so that the stray samples and files can be reunited with the appropriate data sets. Your supervisor has asked you to analyse the sequences and answer some related questions.

A website you need to familiarize yourself with is at the National Center for Biotechnology Information and, within that site, you will be using NCBI BLAST.

BLAST stands for Basic Local Alignment Search Tool. It is a program that compares a query sequence of nucleotides or amino acids (depending on which type of BLAST that is used) against all the nucleotide and amino acid sequences that have been lodged in genetic databases worldwide.

The 'alignment' refers to how the comparisons are made: that is, by aligning your query sequence against all sequences in the database and looking for the best match.

A BLAST search is the standard way for biologists to identify sequences of genes or proteins, or their closest relatives.

The unknown sequences: The unknown sequences will be provided in a separate word document in the bioinformatics assignment folder on the unit site (the same place you found this file).

Assignment instructions and questions -

Follow these steps in order for each sequence separately

In order to identify the unknown sequences provided, you will need to do a BLAST search for each sequence.

Step 1. Go to the BLAST page on the NCBI web site. There are five search options available under 'Basic BLAST': nucleotide blast, protein blast, blastx, tblastn, tblastx.

Step 2. Choose which type of BLAST program you need to run (only consider the first two options). That is, what type of sequence are you submitting - is it nucleotide or protein? (look at the unknown sequence, shown in Step 3, before you make your choice)

Step 3. Copy and paste one the first sequence from the unknown sequences document into the box where it says "Enter Query Sequence" (assume that the sequence is FASTA - which just refers to a format that the program can recognize).?You can give the job a title if you wish, but it is not necessary. However I would recommend naming each sequence as you do them (unknown A unknown B etc) so you don't get confused.

Step 4. You will need to "Choose Search Set". In making this decision, consider the source of your unknown sequence; that is, is it from human, mouse, or some other source? None of these sequences are from mouse or human. Leave the "Program Selection" as 'Highly similar sequences'.

Step 5. Then, click 'BLAST'. It may take a few minutes for your query to be answered. Consider what is happening to your sequence in these few minutes: it is being compared to every known sequence in the databases! - gazillions of them ..

Answer the questions below in the table that was included for the bioinformatics assignment in your prac worksheet handouts. You can print or handwrite as you wish. If you hand write it needs to be legible.

Question 1 - Which type of BLAST search did you choose, and why?

Step 6. Once your BLAST search has returned a result, you will see will see a Graphic Summary of the gene, Descriptions, and Alignments. Hold your mouse over the lower, thinner red line in the Graphic Summary and notice what it tells you above the box. If there is more than one sequence that produced a significant alignment from the Blast search, choose the sequence that you consider has the greatest amount of similarity with your unknown sequence. Notice that the colour of the line in the Graphic Summary represents the alignment score (with red being the highest match, pink is next best, etc) and the length and position of the line represents where the query sequence (your unknown sequence) matches the retrieved sequence. If there is more than one red line chose the top line.

In the Descriptions section, click on the accession number (the number under 'Accession', on the far right of the screen) of the sequence that you think matches (aligns) best with your unknown sequence. (The accession number is a unique identifier given to a sequence when it is submitted to a sequence database.) Again if there is more than one item with 100 % identity chose the top one. This will take you to the GenBank entry for the gene. There are many things listed here and some of them will enable you to answer the following questions:

Question 2 - What is the protein (coded for by the gene for a nucleic acid sequence) and in what is the function of that protein in the cell?

Question 3 - What organism has the unknown sequence come from? Provide the binomial species name (e.g., humans are Homo sapiens) in the correct scientific format. Note that GenBank does not display the correct scientific format.

Question 4 - Is this organism a prokaryote or a eukaryote?

Hint. In the GenBank entry, there is a line that refers to the Organism that the gene comes from. This gives the species name and the various taxonomic groups, or taxa (see Campbell Fig. 1.14) that the organism belongs in, from biggest to smallest, starting with either Eukaryote or Prokaryote and ending with the genus.

Now, let's consider the sequence of the gene and its translation product, that is, the protein. If you haven't already done so, read the Supplementary Information B (below) on the Klp1 gene and how cDNA sequences are derived from mRNA. The same is true for any nucleotide sequences you are now looking at.

Step 7. If it is a nucleotide sequence click the blue-highlighted "CDS" (coding segment/sequence), which will shade, in brown, the sequence of nucleotides that codes for the protein.

Question 5 - What is the STOP codon for the gene? Remember, there are 3 alternative stop codons; give the nucleotide sequence (as it appears in the coding sequence) for the stop codon used by this gene.

Note: you will not be able to answer this question if the sequence is an amino acid sequence so you can leave it blank in those instances.

Question 6 - How many nucleotides are there in the coding sequence for this protein? Hint: use the numbers in the left-hand column to help you count, or the numbers next to CDS and remember the coding sequence is comprised of codons which are 3 nucleotides each which will code for one amino acid. Therefore the total number of nucleotides in the sequence must be divisible by 3.

Question 7 - How many amino acids are there in the entire protein (number of amino acids in the translated sequence?

Supplementary information: A. The example sequence (the Klp1 sequence) that was used in the Intro to Bioinformatics lecture had a GenBank entry described as "C. reinhardtii mRNA for kinesin-like protein". Why does the GenBank entry show DNA sequence (it contains only A, C, G, T) and not RNA sequence (there is no U)? The reason is that mRNA from Chlamydomonas reinhardtii was isolated for the sequencing of this gene, but it was first 'reverse transcribed' into complementary DNA (cDNA). The sequence of cDNA is shown. This will also be relevant when you do the assignment. To know more about reverse transcription and why biologists do it, see Campbell 10e concept 20.2. You will learn more about cDNA and reverse transcription in the lectures on biotechnology.

Attachment:- Assignment Files.rar