Tutorial: PCR Analysis

We will use the PCR analysis code to amplify a promoter region of a Drosophila hsp70 gene.

  1. Open the file Hsp70 protein found in the Tutorials folder. You will see Figure 2.78.
    Figure 2.78: hsp70 protein construct
  2. The arrow below the DNA is the actual transcript for this Drosophila heat shock gene. We want to select the 300 nts upstream of this transcript to amplify, which we suspect has an important promoter element. Double-click on the arrow to select the corresponding DNA segment. This will select nucleotides 1617-3677, as indicated in the bottom left corner of the construct window.
  3. Switch to sequence view (Construct > Display > Display Sequence). The same region is selected. Using your mouse, click and drag “upstream” starting at the first nucleotide before the coding sequence, 1616. Drag approximately to 1317 and release the mouse button. You can fine tune the selection by holding down the shift key while selecting a new starting point in the sequence. Keep trying until you have selected nucleotides 1317-1616. Keep this selection and switch back to the graphics view (Construct > Display > Display Graphics).
  4. Select Tools > PCR Analysis to bring up the PCR dialog. You will see something like Figure 2.79. If you click on an item in the Parameters list, you will see a description of that item on the right side of the window. Feel free to explore the parameters that you are allowed to set. Those parameters that say “optional” can be left as they are, or you can fill in values for them. For now, set the 5′ and 3′ allowable ranges to 75 and press the Generate Primers button.
    Figure 2.79: PCR setup panel
  5. This will bring up Figure 2.80. There are 5 possible primers that meet the criteria set in the original parameters. The bottom of the window presents some statistics on how all possible primer pairs were filtered to generate the list shown in the window.
    Figure 2.80: Selecting a primer pair
  6. Select the middle primer pair (1293-1312, 1762-1781) and press the Add Primer button. If you want to, you can add multiple primers to the construct, but for now, just add the one. After you have added the primer, press the Done button.
  7. You will see Figure 2.81, which shows the two primers. Clicking on either primer will select the primer pair. Once you click on the pair, you can get information by pressing command-I (Mac) or control-I (Windows), or choosing the Construct > Get Info menu item. The results of the Get Info window are shown in Figure 2.82. This provides all the details of how the primer was actually generated from the original sequence.
    Figure 2.81: Marked primers
    Figure 2.82: Primer information
  8. You can double click on the primer pair (either of the primers will work) and it will select the DNA amplified by the primer pair. You can now copy this sequence to another window (or another application) to work with it in more detail.

1. GCK uses Primer3 PCR code developed at the Whitehead Institute for Biomedical Research and used with permission. This code is Copyright (c) 1996, 1997, 1998, 1999, 2000, 2001, 2004 Whitehead Institute for Biomedical Research. All rights reserved. The algorithm for this software is published: Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386.

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