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To design primers, we used software called CLC DNA Workbench. The first step in this process, after we got our raw data back from our mentor, Dr. Roberto's, lab is to create a consensus sequence of the raw data. The raw data is a list of various short overlapping segments of base pairs from the DNA. To create a consensus sequence, we arranged several segments of DNA by matching them up according to base pairs. We then take the most common base pair from each part of the segments and form a sequence from them. For example: if three of the segments we were examining had a G, and one had a C, we would use a G in the consensus sequence. Consensus sequences can also be used to combine several overlapping segments into one larger segment by finding the places where the segments overlap and creating a sequence that eliminates the overlap. To do this we use a software called CLC DNA Workbench. Then we BLAST our consensus sequence to find what it is similar to. Ours is most similar to Thermus Brochianus, Thermus Aquaticus, and Thermus Thermopholus. Then we locate the already sequenced polymerase genes for these similar species and use them to design primers. Hopefully, the genes will be similar enough that the primers that work on these polymerase genes will also work on our polymerase gene. We then put these sequences into a program called Primer3 to design our primers: http://frodo.wi.mit.edu/primer3/. We ordered our primers from Integrated DNA Technologies: http://www.idtdna.com/catalog/CustomSynthesisandPurification/Page1.aspx. ) We use these options when using Primer3 (click to increase size). First, we paste in the consensus sequence of the similar species. We then changed the size range to 2000 – 2500. This size range would allow us to get most of the polymerase gene. We left all other settings blank or at default and clicked pick primers. Scroll to the bottom of the page to see the primers. ) The results for our primers (click to increase size). The next step is CLC Procedures. |
