Tutorial: Working with Generic Constructs

There are times, especially when you are just beginning to work with a piece of DNA, when you might know the restriction map of a cloned fragment of DNA but you might not yet know the sequence. Such generic constructs can be created in GCK and the segments manipulated just like any piece of DNA whose sequence is known.

  1. Start GCK. You should see an untitled Construct window as the active window. To create a generic construct of 5000 nucleotides, choose Construct > Insert Ns… ( command-H/ctrl-N ). This will bring up Figure 2.58. Enter 5000 and press the OK button.
    Figure 2.58: Inserting Ns
  2. You should now have a linear construct containing 5000 Ns. In the graphical view, it will simply appear as a horizontal line. Choose Construct > Display > Display Sequence to view the sequence as a series of Ns. Now you need to place restriction sites at specific locations in the construct.
  3. Choose Construct > Features > Place Sites… ( command-J/ctrl-J ). You will see Figure 2.59. This is very similar to the mark sites dialog you saw previously (Figure 2.8). You select enzymes in the top list and press the Add button to move them to the bottom, Sites to Place , list. For this tutorial, choose BamHI and EcoRI.
    Figure 2.59: Place Sites Dialog
  4. The next step is to define the locations of each of the enzyme sites you will be placing into the construct. This is done by first selecting an enzyme in the bottom list and then defining the locations of the sites for that chosen enzyme. Select BamHI in the bottom list and then press the Locate button on the right. This will bring up the locate sites dialog shown in Figure 2.60. This dialog gives information about the selected enzyme and then lets you define the site locations. Type in 200 in the Starting at position text box and press the Add button to add this location to the list of sites to place – it will appear in the list of “instances” at the right. Next type in the position 1000 and add it to the list, and then add 3456. You should have these three locations in the list on the right. Once you have done this, press the Done button because you are done entering locations for BamHI sites.
    Figure 2.60: Locate Sites Dialog
  5. Select EcoRI in the list (Figure 2.59) and then press the Locate button. In the locate sites dialog that appears (Figure 2.60), enter 2000 and 4000 as the EcoRI site locations. You have now defined the enzymes and their sites so press the OK button to actually place those sites.
    Figure 2.61: A Placed Site (Sequence View)
  6. In Figure 2.61 you can see that the BamHI recognition sequence has actually replaced some of the Ns. This is therefore a “real” site that can be cut with the enzyme and used to produce fragments from this DNA.
  7. Choose Construct > Display > Display Graphics . This will bring up Figure 2.62. You can now select fragments and use them in cloning steps as was done for fully sequenced DNAs in previous tutorials. As more sequence information becomes known for this construct, you can use the real sequence to replace the Ns in this generic construct.
    Figure 2.62: Placed Sites (Graphic View)

This concludes this tutorial. You can close the open window (don’t save changes – you will not need this file any more).

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