The first step in my experiment of cloning and tagging PCH proteins in Dicty (Dictyostelium discoideum) is to isolate the DNA sequence that codes for the production of these proteins. To isolate this sequence, I had to design primers that would signal the cut off this specific DNA sequence for the enzyme used. Due to working with this technique of designing primers for the first time, I received basic background information on how it works.
When a DNA strand is ready to replicate itself, helicases unzip the DNA. This results in two single strands of DNA, one going 5’ to 3’ and the other running the opposite way of 3’ to 5’. Primers that are specific to certain sequences bind with specific parts of the DNA, matching G with C, A with T, and vice versa. There are two primers that are involved, a forward primer and a reverse primer. In a forward primer, the primer reads the DNA strand left to right, much like we read a book. This is due to the fact that the enzymes we are using synthesize DNA from the 5’ end to the 3’ end. In contrast, our reverse primers make reverse compliments of DNA, reading them from right to left. This is because the 5’ end is on the right, meaning the sequence must start from the 5’ end. This process of primers starting the sequence is the first step in annealing. Annealing, which is done at a specific temperature, ends when an enzyme finishes the sequence that the primer has started. I will use the process of Polymerase Chain Reaction (PCR) to hopefully replicate many of this DNA sequence for PCH proteins.
In my experiment, I do not want the entire strand of DNA to be recoded. I want to isolate the genes that code for a specific member of the PCH protein family. To isolate this specific gene, I needed to design primers that only acted on this gene. The primer needed to be at least eighteen nucleotides long and needed to be at a certain temperature to begin the annealing process. Because I am using two primers in my experiment, I needed the annealing temperature of the two primers to be within 5 degrees Celsius. Another problem is that my primers may actually bind to each other. To test this, I ran a hetro-dimer test, with a result of negative five or greater meaning that the two primers would not bind to one another. I also needed to make sure that these primers only bind to m specific gene and not to another. This test was done using BLAST (see final link), in which I compared my primers with other sequences of DNA that it may also attach to.
To design these primers, I used two different online databases to determine the optimal sequence of genes for my primers (see links below). I used the first website to find out what the gene sequence was for my PCH protein. Once I found the sequence, I went to the second link to find out what my forward and reverse primer DNA sequences should be. To do this, I started with the first eighteen nucleotides and found their forward compliment used analysis, hairpin, and self-dimer tests. The analysis of my eighteen nucleotides showed me the melting temperature and the percentage of Cs and Gs in my sequence. Optimal conditions for my primers would be for them to melt between fifty-five and seventy degrees Celsius and to have about 50% of the sequence contain Cs and Gs. This is because C-G bonds involve three hydrogen bonds compared to two in A-T bonds, making the primer more stable. If this passed I then looked at the hairpin and self-dimer tests which displayed delta G outputs for my chosen nucleotides. The nucleotides could work if I had a delta G output of greater than negative five. I then BLASTed the two primers to see if these specific primers would bind to any other parts of my dicty DNA. Doing so would compromise my experiment. If I found a forward and reverse primer that satisfied all of these criteria, I then ran a hetero-dimer test to see if the two primers would bind to each other or not.
Links:
What are PCH proteins?
ReplyDeleteI noticed that you wrote about different things that could possibly happen such as: binding together or binding to else...are there any articles describing previous research techniques that have been used to avoid this? I also noticed that you mentioned the hetero-dimer test and the BLAST test...I'm assuming that these test ensure that your results are accurate?
ReplyDeleteHey Casey,
ReplyDeleteIt sounds like you've been put through a pretty intense first week of research! I am unfamiliar with designing primers myself. Do you know what is the significance of the test for hairpin turns?
This process sounds very interesting! I reminds me of what I had to do for making my own primers for a project I recently did in biochem. For me it was hard to find a sequence for to build the primer, with your website did it work for just proteins or could you use plants too? Good luck with your PCR!
ReplyDeleteWhat is the difference between testing for hairpins and testing for heterodimers?
ReplyDelete-kc
Casey,
ReplyDeleteFor just beginning this week you have sure learned alot about designing primers as well as their usefulness in PCR. It like how detailed you have made your blog. It was interesting to read the process as it went through your mind. I hope that they work for you!
Dr. Cooper and Ty: testing for hairpins is testing to see if one primer interacts with itself. A heterodimer test is used to see if two primers will interact and/or bind with each other.
ReplyDeleteFelicia: I didnt see any plants on my list, but i wasnt looking for them either. However, one of the primers is used in protein synthesis in humans
Jake: Good question Jake! PCH proteins are a family of proteins found in cells that are involved in cell movement and membrane bending.
Cara: Theres really no way to avoid two primers to interact with each other or with themselves. The only way to avoid it is by choosing primers and running them through the tests to see if they will or not. If they do, I had to pick another primer. A heterodimer test is a test to see if the two primers i am using will bind to one another. A BLAST is a database that lets me type in the primer sequence I am using and it will let me know what other DNA sequences it will attach to. For example, one of the primers I am using in my research codes for PCH proteins in dicty, but is also used in humans as well. I used BLAST to make sure the primer I chose did not connect to any other DNA sequence other than the one I wanted for dicty. Good questions!
Casey,
ReplyDeleteBoth true about hairpins and heterodimers. But you also tested for homodimers. What are those?
-kc
I tested for self-dimers but not homodimers. Self-Dimers are another test to see if the primer will have intermolecular interactions with itself instead of the DNA sequence we want it to attach to. The lower this delta G, the more it binds to itself and not the DNA sequence, which produces less of the desired yeild. Again, a score of -5 or higher is considered a good primer to use.
ReplyDelete