Sequence Analysis

  1. Download the file LacZ.gb and open in a text editor.
    • This is a Genbank format file that contains the sequence following the word ‘ORIGIN‘ and terminating with ‘//‘.
    • Prior to the sequence is a batch of descriptive information including references, organism and database cross-reference identifiers. While these don’t mean much to you, the appropriate database within Genbank can be queried to reveal more information about the sequence.
  2. Download the file LacZ.fasta and open in a text editor (NotePad).
    • Notice the simple structure of the fasta file beginning with the ‘>’ and description of the sequence.
    • This is a DNA sequence. But DNA is usually double stranded! We can assume the sequence of the second strand because it will be complimentary to this one.
      • By convention: we know that this sequence is 5′ → 3′
    • This text contains a portion of the E. coli genome that includes a gene called LacZ.
    • This file does not contain any annotation to indicate where the gene sequence actually begins or ends.
  3. Launch UGENE and open both files. They will appear on the left side “Objects” pane.
    • The default display automatically shows the reverse compliment of the DNA strand and all 6 Open Reading Frames (ORFs).01_ugene
    • To simplify the view, click on the ‘C‘ to remove the complimentary strand (look at the cursor in the image)02_ugene
  4. Count the ORFs :
    • Find ORFs by right-clicking on the sequence and select “Analyze → Find ORFs
    • Default setting looks for ORFs on both strands with a minimum length of 100 nucleotides
    • The Open Reading Frame here is defined as something beginning with initiation or start codons from the Standard Genetic Code (ATG) and two additional alternative start codons (TTG & CTG) that is terminated by any one of the three standard stop codons (TAA, TAGTGA)
    • Selecting Preview will provide the amount of possible ORFs fitting these criteria.011_ugene
  5. Double click on the LacZ.gb in the Objects panel to activate the view.
    • This file now shows the same sequence with information about the DNA
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    • Expand the various features in the Annotations pane at the bottom to explore the sequence features.04_ugene
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