Tutorial: Cloning a DNA Segment and Silent Mutations

Our goal in this tutorial will be to take the construct we have been working with in previous tutorials and use it to receive a new segment of DNA. In doing this you will learn about copying and pasting segments of DNA and how to keep track of the history of your construct.

    1. Start GCK and open the file construct#5 that you saved in the previous tutorial. If you do not have this file, you can start with construct#5 from the tutorials folder provided.
    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. The first thing to do is mark the BamHI sites in hsp70 . Make sure that the window with hsp70 is in front and then choose Construct > Features > Mark Sites [or press command-M (Mac) /ctrl-M (Windows)]. 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 2.8). Select to display new sites as enzyme names. Press OK to mark the sites. You will see Figure 2.22.
      Figure 2.22: BamHI sites marked on hsp70
    4. To cut out the segment we are interested in we need to address the problem of the middle BamHI site. In the lab, you could do partial enzyme digests and obtain the fragment containing the entire coding region by elution of the fragment from a gel. You can also select this entire region in GCK by dragging with the mouse from the first (left) BamHI site in the DNA to the third BamHI site. This fragment can be copied and pasted into the vector. An alternative is to eliminate the middle BamHI site through a silent mutation. This mutation will eliminate the BamHI site without interfering with the coding capacity of the DNA.This is the approach we will take in this tutorial.
    5. To make a silent mutation you first need to specify the reading frame to which you are referring. To do this, select the single region in the hsp70 construct by clicking on it once. Now choose Construct > Features > Remove Sites by Silent Mutation. You will see the familiar dialog shown in Figure 2.23. Select the BamHI enzyme in the list on the left and press Add -> to bring it to the right list of Enzymes to try . Click OK. This will bring up Figure 2.24. Click on the BamHI name in the list to select it (if there were more than one BamHI site, you could choose any one or more of them). Now press OK.
      Figure 2.23: Select Enzyme Sites to be Removed
      Figure 2.24: Selecting the Site To Remove
    6. This will bring up another dialog containing a list of the different possibilities that could be used to remove the BamHI site without altering the coding information in the DNA. This is shown in Figure 2.25. The original sequence is shown in the first column and the possible changes are shown in the second column. The actual changed nucleotide is shown in lower case. Select the first item in the list (as shown) and then press the OK button.
    7. You should now see the hsp70 construct with the middle BamHI site missing. The actual nucleotide sequence has been changed as specified in Figure 2.25 with the changed nucleotide being indicated in lower case in the actual sequence. [If working with the demo version of GCK, saving is disabled, simply continue to step 8 below]. If working with a ‘Trial Version’ or the ‘Full Version’ of the application, choose File > Save As… , name the file myhsp70_Bam- , and save it in your own folder – please do not replace the tutorial files provided because others might want to use them later. You will need this file again in Tutorial 10: Making Illustrations.
      Figure 2.25: Selecting a Silent Mutation
    8. You are now ready to proceed with the cloning. Click in the DNA segment at the left BamHI site and drag the mouse cursor to the right until you have selected all the DNA between the two BamHI sites. The entire segment should be highlighted.
    9. Choose Edit > Copy [or press command-C (Mac) /ctrl-C (Windows)] to copy this segment to the clipboard. Make the construct#5 window the active window either by clicking in the window with the mouse or by selecting it from the Window menu.
    10. You now need to tell GCK where it is that you would like to paste the BamHI fragment that is now 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. You are finally ready to do the actual cloning step. Choose Edit > Paste . You will be shown a dialog warning you that the editing operation you are about to perform (pasting in a fragment) will disrupt a region in the current construct (the beta-galactosidase segment that spans the polycloning site). Press OK to that dialog. You should see Figure 2.26.
      Figure 2.26: Pasted hsp70 Fragment
    12. Notice in this figure that the coding region from hsp70 is intact and displayed 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. [If working with the demo version of GCK, saving is disabled, simply continue to step 14 below]. If working with a ‘Trial Version’ or the ‘Full Version’ of the application, choose File > Save As…, name the file myconstruct#6 , and save it in your own folder – please do not replace the tutorial files provided because others might want to use them later.
    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 (in memory) and you have a segment of DNA selected, pasting will replace the current selection with what we paste into the construct from the clipboard. This is just like replacing selected text in a word processor with the contents of the clipboard. You will see Figure 2.27. 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.
      Figure 2.27: Special Paste
    15. [If working with the demo version of GCK, saving is disabled, simply continue to step 16 below]. If working with a ‘Trial Version’ or the ‘Full Version’ of the application, choose File > Save As… , name the file myconstruct#7, and save it in your own folder – please do not replace the tutorial files provided because others might want to use them later. 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 in a real experiment. You will be able to use these constructs and GCK to predict gel patterns as detailed in Tutorial 9: Running Gels and Orientation Analysis. This demonstrates how easy it is to replace DNAs and to generate constructs with inserts in either orientation.
    16. Before ending this tutorial let’s see how GCK behaves 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.
    17. With the insertion point in the DNA at the selected site, choose Edit > Paste [command-V (Mac) /ctrl-V (Windows)]. This will bring up a ligation dialog, as shown in Figure 2.28. 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 – 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. 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. There is a shortcut to this process using Edit > Special Paste, which is discussed in detail in “Special Paste…”. For now, press Cancel.
      Figure 2.28: Ligation Dialog
    18. Close construct#6 and hsp70_Bam- but do not save any changes. You may continue with the next tutorial now, or quit the program.

    1. Note that this sequence has been derived from an hsp70 gene from Drosophila. The original sequence has been modified for use in this tutorial. Do not use this sequence as a real hsp70 sequence.

    2. You might notice that the selection “pauses” at the location of the former middle BamHI site. This is because the changed nucleotide has a different color and represents a “boundary” in the sequence. Selections in the graphical view occur between boundaries.

    3. This alert is shown to warn you of editing operations that you might not be aware of. If you are pasting into a site that is part of a “feature” in the construct, the pasting operation will disrupt that feature. Therefore it needs to be removed to prevent any confusing or ambiguous annotations from remaining.

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