|
1. Start GCK and open the file construct#5 from the tutorials folder.
2. Open the file called hsp70 from the tutorials folder. This file contains the gene we will be using as the source of the fragment being cloned. Notice that there is a single region indicating the open reading frame. We will be cloning a segment containing this region into the BamHI site on construct#5.
3. Make sure that the window with hsp70 is in front and then choose Construct > Features > Mark Sites. Select BamHI in the left hand list and press the Add -> button to add this enzyme to the Enzymes to mark list on the right (see Figure 8). Select to display new sites as enzyme names. Press OK. You will see Figure 23.
|
|
Figure 23: BamHI Sites Marked on hsp70
|
4. To cut out the segment of interest in we need to address the problem of the middle BamHI site. In the lab, you might do this by partial digests. An alternative is to eliminate the middle BamHI site through a silent mutation, which will eliminate the BamHI site but not change the coding information.
|
|
Figure 24: Select Enzyme Sites to be Removed
|
5. To make a silent mutation you first need to specify the reading frame to which you are referring. Select the single region in the hsp70 construct by clicking on it once. Choose Construct > Features > Remove Sites by Silent Mutation. to bring up Figure 24. Select the BamHI enzyme in the list on the left and press Add ->. Click OK. This will bring up Figure 25. Click on the BamHI name in the list to select it and press OK.
|
|
Figure 25: Selecting the Site to Remove
|
6. This brings up a list of changes that could be used to remove the BamHI site without altering the coding information, as shown in Figure 26. The original sequence is shown in the first column; the possible changes are shown in the second column. The actual changed nucleotide is in lower case. Select the first item in the list (as shown) and then press the OK button.
|
|
Figure 26: Selecting a Silent Mutation
|
7. You will see the hsp70 construct with the middle BamHI site missing. The actual nucleotide sequence has been changed as specified in Figure 26 with the changed nucleotide being indicated in lower case in the actual sequence. This construct is in your Tutorials folder saved as hsp70(Bam-).
8. You are now ready to proceed with the cloning. Click in the DNA segment at the left BamHI site and drag the mouse to the right until you have selected all the DNA between the two BamHI sites.
9. Choose Edit > Copy to copy this segment. Make the construct#5 window active by clicking with the mouse or choosing it from the Window menu.
10. You now need to tell GCK where it is that you would like to paste the BamHI fragment currently in the clipboard. To do this you must position the insertion point at the BamHI site in construct#5. The easiest way to do this is to click on the site marker text, BamHI. Do this now.
11. Finally, choose Edit > Paste. You may see a dialog warning you that the editing (pasting) operation you are about to perform will disrupt a region in the current construct. Press OK. You should see Figure 27.
|
|
Figure 27: Pasted hsp70 Fragment
|
12. Notice in this figure that the coding region from hsp70 is intact and that the size of the construct shown in the center of the window is adjusted to reflect the newly inserted fragment. Because a BamHI fragment was pasted into a BamHI site, both of the BamHI markers have been restored.
13. This construct is saved as construct#6 in the Tutorials folder.
|
|
Figure 28: Special Paste
|
14. Make sure that the blue inserted DNA segment is still selected and then choose Edit > Special Paste. Since the hsp70 fragment is still on the clipboard and you have a segment of DNA selected, pasting will replace the current selection with the contents of the clipboard. This is just like replacing selected text in a word processor. You will see Figure 28. Check the Invert Sequence check box and select the Leave Ends Alone radio button. Press OK. This will replace the currently selected hsp70 segment with an inverse version of the same DNA -- i.e., it will be flipped over.
15. This construct is saved as construct#7 in the Tutorials folder. Since cloning the hsp70 fragment into the BamHI site has an equally likely chance of occurring in either orientation, these constructs (construct#6 and construct#7) represent two possible outcomes of a real cloning experiment. This demonstrates how easy it is to replace DNAs and to generate constructs with inserts in either orientation.
16. What happens if you try to paste the BamHI fragment into a site other than a BamHI site? The fragment is still on the clipboard so all you need to do is define a different insertion point in the construct to be the target for the pasting operation. To do this, click on any symbol in the construct to select that site.
|
|
Figure 29: Ligation Dialog
|
17. Choose Edit > Paste. This will bring up a ligation dialog, as shown in Figure 29. Because the fragment ends are incompatible (the selected site in construct, BamHI in clipboard), GCK will not let you paste the fragments together (ligate them). The sequences shown in this dialog represent the junction shown in the top left corner of the dialog -- in this case, the left DNA sequence is the result of the selected cut in the vector and the right DNA segment is the result of the BamHI cut fragment in the clipboard. The arrows indicate the actual cut sites and illustrate the staggered ends. Note that the OK button is disabled because the operation is not biologically possible -- the ends are incompatible. To adjust the ends you can drag the arrows with the mouse until you have compatible ends. As soon as the ends are made compatible, the OK button will become enabled and you will be asked to deal with the other junction between the fragment being pasted and the target sequence. Press Cancel to close the dialog without pasting.
18. Close construct#6 and hsp70(Bam-).
|
