The Attractin Gene

This week will be very productive, as I have numerous tasks to complete in the laboratory! Five sets of primers (short nucleotide base chains that correspond and bond to each strand of a sample of template DNA in order to amplify the regions between the two primers) arrived last Friday. These primers are spaced throughout the attractin gene, which is a large gene, so that I can determine whether most regions of the gene or simply the one I previously sequenced in a population of mice from Kenzin is unrelated to coat color variation; I can also use these primers to determine whether variation in the attractin gene in the Carrizozo population of mice is related to coat color variation. In order to begin sequencing these five other regions of the gene in both the Kenzin and Carrizozo mouse populations, I must determine the appropriate parameters for the PCR reaction (a method of amplification of a specific region of a gene). Thus, I am running gradient PCR reactions using DNA from one light agouti, one dark agouti, and one melanic (dark) mouse from Kenzin, NM, with each of the five sets of primers. Gradient PCR reactions contain a variety of primer annealing temperatures (temperatures at which the primer binds to the complementary strand of DNA) to determine the optimum temperature that obtains the desired results of DNA product. The results of the gradient PCR then determine what additional alterations are necessary for the PCR to obtain the most desired product and lack nonspecific product. For instance, the first gradient PCR that I ran today located two optimum annealing temperatures at 49 and 51 degrees Fahrenheit; the higher temperature is best for avoiding nonspecific product because higher temperatures create greater instability and thus DNA regions most complementary to the primers will amplify most successfully. However, there was nonspecific product (product that was not part of the expected region of the gene) at both temperatures, so I will also alter the MgCl2 (Magnesium chloride) concentration; a lower MgCl2 concentration acts the same as a higher annealing temperature, resulting in more specific, desired product. Additionally, I will shorten the annealing time so that the larger, nonspecific product does not have time to form. I will continue altering the parameters of the PCR reaction for all five sets of primers until I locate the optimum conditions for each set, at which time I will begin the steps necessary to sequence the various regions of the attractin gene in the Kenzin and Carrizozo populations of mice.

Last week, I received more data (DNA sequences) to analyze. Thus, in addition to optimizing the PCR reactions, I will be cleaning and analyzing the data (as described in my previous blog post on Friday, February 19, 2010). I look forward to examining this data in search of more results and will post any results I do obtain. I also attended lab meeting today and learned about an experiment being conducted under Professor Nachman concerning the genetic basis of reproductive isolation in mice.

This week has already been busy and will only continue to be eventful and productive. I am eager to optimize the PCR reactions and actually begin to sequence more regions of the attractin gene. Each experimental step I perform now will hopefully lead to more successful results in the coming weeks!