Lab 11

Overview

To receive full credit for this lab, you must get through all questions other than those labeled after the "Extra time?" prompt.

Download printable copy.

Because it will be easier to properly predict genes for a prokaryote than a eukaryote, we are going to start with a prokaryote, such as E. coli. But since the full genome is rather large, we will also start with a smaller self-replicating DNA molecule known as a plasmid, specifically plasmid pACYC184.

1. Go to the NCBI database and search for pACYC184 in the nucelotide database; we are specifically interested in the one labeled as "Cloning vector pACYC184".

   Question: How many nucleotides does this sequence have?

   Question: What is its accession number?

   Question: What are the first 10 nucleotides reported in its representation? (To be fair, this is actually a circular molecule, so the "start" is only by convention.)




2. Next, go to NCBI's ORF finder and enter the accession number for pACYC184, and have it compute all ORFs using the default parameter settings.

   Question: How many such ORFs are found?

   Question: How many nucleotides are in the longest ORF?

   Question: At what nucleotides does the longest ORF start and stop?




3. By default, it reported all ORFs having length 75nt or longer. Redo the search with minimum ORF length of 150nt.

   Question: How many ORFs have 150nt or more?




4. Return to the search page. There is an option to "Ignore nested ORFs". Redo the search with this option and minimum length of 150nt.

   Question: How many orfs are reported?




5. Compare the results when including nested ORFs and the results when excluding them. Locate at least one specific ORF that is excluded in the latter search.

   Question: What is the start..stop of such a nested orf that was excluded? What is the remaining ORF in which it was contained?




6. Click on the longest ORF to examine its details. Notice a box to the left that by default shows its amino acid sequence.

   Question: What are the first four amino acid characters?




7. You can switch to see the underlying nucelotide sequence by clicking on the "Display ORF as..." label.

   Question: What are the first 12 nucleotides?

   Question: Which of the stop codons ends this ORF?




8. Not every ORF is necessarily a gene. One way to suggest that an ORF is a gene is by comparing its sequence to a database of known genes from other genomes to look for similarity. BLAST is a popular such tool (and we will soon explore the underlying algorithm it uses for sequence alignment). The NCBI ORF Finder conveniently offers a button to perform a BLAST search for a selected ORF. (In fact, there is a "BLAST" button and a "SmartBLAST" button.) Let's use the SmartBLAST button.

   Question: What conclusion is suggested by a SmartBLAST on this ORF?




9. Let's go an examine the second longest of the identified ORFs (after removing nested orfs).

   Question: How many nucleotides are in this ORF?

   Question: At what nucleotides does this ORF start and stop?

   Question: How would you interpret the fact that its start index is larger than its stop index?

   Question: What conclusion is suggested by a SmartBLAST on this ORF?




10. Go back to the original database in which we found this plasmid. Within that view, there is a section labeled "FEATURES". Notice that two of those miscellaneous features are described as genes.

    Question: give the start..end indices and descriptions for the two genes.

    Question: Which of these corresponds to the longest ORF that we examined earlier?

    Question: Can you find an ORF that corresponds to the other of these identified genes?

    Question: What if we remind you that this was actually a circular molecule? Can you find a pair of ORFs that are reported by the ORF finder that together form this gene?

Extra time?

Begin a similar such analysis on the guinea pig mDNA, to see whether the longest ORFs correspond with identified genes.