Simply Cloning - Supplement 3 - pDRAW32

Illustration on how to build vector maps and design primers in pDRAW32 - free DNA editing program from AcaClone Software. Narration Script: pDRAW32 is a free DNA analysis software from the AcaClone Software. It does not have all the features of Vector NTI, but it is pretty good for simple DNA map building and primer design tasks. In this chapter I am going to use pDRAW32 to build maps of pSAT6-MCS and pSAT6-Bar and to design primers for amplifying the bar gene in PCR. To get started, I go to PubMed Nucleotide and find sequences of pFGC5941, which I will use as a PCR template for bar gene, and a map of pSAT6-MCS, which is the vector I will clone into. Then I launch pDRAW32, go to File - New - Enter New Sequence, and insert here the sequence of pSAT6-MCS. For that I go back to the web browser, scroll down, select the sequence, copy it, go back to pDRAW32 and paste it. Now I am going to add features to this sequence. I go to File - Edit and click on “DNA name, properties and annotations“, where I can add the map features. I get the features of pSAT6-MCS from the PubMed file. I switch again to the web browser window, scroll up and select the Feature Table view. You can see a list of pSAT6 features with their coordinates. Let’s transfer them to our map. For each feature I put its name, start and end position, orientation and type. Then I press on ADD. After all the features have been added I could check them with preview button. Then I switch to the DNA information tab. Here I insert the name of the plasmid and indicate that it is a circular DNA. Now it looks like a pretty map of pSAT6-MCS. Let’s save it. Notice, that if you want to see the restriction sites on the plasmid map, you have to close and open pDRAW32. Now I am going to build a pSAT6-Bar map by pasting the sequence of the bar gene into the pSAT6-MCS. Let’s first save this map as pSAT6-Bar. In pDRAW32 we need to separately edit the plasmid name, because it is not automatically imported from the file name. Now, let’s go back to the PubMed Nucleotide and locate the sequence of the bar gene. When we scroll up and look at the annotations we could see that the sequence of thebar gene is located on the reverse strand in the positions 373 - 924. To make life a little simpler, I will use PubMed Option called Change Region Shown. I will insert here the coordinates of the bar gene and click on Update View. Now in my web browser I have the sequence of bar gene that I am going to copy. Before pasting this sequence into pDRAW32 I want to change its orientation by using a utility called Sequence Manipulation Tool. I paste here my sequence, select Reverse Compliment option and calculate the sequence. Here is the sequence of the bar gene, starting with the ATG and ending with the stop codon. I am going to copy it and hide this window for now. Editing a plasmid sequence in pDRAW32 is somewhat not trivial, but it can be done. First, I open a Sequence View window and locate the sites of XhoI and BamHI. Here we have CTCGAG, which is XhoI, and here we have GGATTC, which is BamHI. Please notice that those two restriction sites are located on lines beginning with nucleotides 1301 and 1351. Now I open an Edit Sequence window, locate XhoI site on the line 1301 and introduce a line break after it. Then I go to the line 1351, locate the BamHI site and introduce a line break before. Now I am going to remove the piece of Multiple Cloning Site between XhoI and BamHI and paste there bar sequence. Here on the map you can see a fragment that has been inserted between XhoI and BamHI sites. I am going to annotate this fragment as the bar gene. For that I will go to Edit - DNA annotations, and add a new feature, which would start in the position 1345 (right after the XhoI site) and would end in the position 1897 (right before the BamHI site). Now, let’s design the PCR primers. With a plasmid map at hand it is quite easy. I am going to open a Sequence View window, hide all the ORFs, scroll down to the XhoI site, select 20 nucleotides right after it, and copy them with Control C. Then I open a Sequence Manipulation Tool and paste the sequence with Control V. I add six random nucleotides at the 5’ end, select a restriction site, and hit Calculate. This is the primer sequence that I will send to an oligo synthesis company. Similarly, for the reverse primer, I will select the last 20 base pairs of bar, paste them into the Sequence Manipulation Tool, add a 5’ overhang and a restriction site, check Inverted button and hit Calculate. Here I have shown you how to build plasmid maps and design PCR primers using some basic features of pDRAW32 .