With the help of SFM or Sailfin molly fish as an emerging organism, we can have a better understanding of the theory of evolution. Some specifications such as the environmental impact, population biology and mating behavior can be revealed by studying the Sailfin molly fish. The “Guppy” genome is the closest relative of the sailfin molly since annotated. However the sailfin molly genome has to be annotated and not been done yet. In order to annotate sailfin molly genome we would need to create a proper polymorphic marker and some websites will help us to accomplish our goal.
Creation of polymorphic PCR marker is needed for studying the population and paternity, thus it will provide us to have a perfect understanding to annotate the SFM genome. http://www.ncbi.nlm.nih.gov/ , or the websites of The National Center for Biotechnology Information, National Institutes of Health, National Library of Medicine are perfectly controlled and monitored by the government of the United States, thus they could be a perfect source of information for annotation of the SMF genome.
Methods and Materials
Creation of the primer for the PCR reaction would be the first step. The primer blast enabled us to locate short sequences in the DNA. The DNA blasting gave us the opportunity to find the microsatellites and thus we could design the primer.
The second step is the extraction of DNA, which was done by taking a piece of SFM tissue, which was already preserved in ethanol. Then it was squished by hand and washed with the help of ATL, PBS, and proteinase K. For 90 minutes this mixture was incubated in 56OC, until it was completely lysed. The mixture after incubation was mixed with Buffer EtOH and AL and fully mixed by vortexing, and then it was centrifuged by DNeasy Mini Spin. The liquid solution was discard after being centrifuged. The centrifuging process was repeated by transferring the pin to a new tube and addition of Buffer AW1. This process was repeated one more time after the tube was discard, pin was transferred to a new tube and Buffer AW2 was added, and it was centrifuged again.
The next step included repeating this process one more time, but this time the pin was transferred to a 1.5 mL micro-centrifuge tube Buffer AE solution. It was incubated at the room temperature for one minute, and then centrifuged for one more time. Thus the purity and concentration of the extracted DNA was estimated.
Polymerase chain reaction (PCR) and analysis was the last step, which consists of three critical steps. The steps are Denature, Anneal and Extend. The first step is Denature of the primers, the DNA template and dNTPs at 95OC. Second step includes annealing, as we let the mixture to cool down to 60OC, thus the primers can hybridize with signal strand of DNA. And the final step is extending. Included the increasing of the temperature of the mixture up to 72OC, which is the optimum temperature for Taq polymerase. Though in order to stop the reaction we had to increase the temperature up to 95OC. The PCR reaction took place in PROMEGA Go Taq Green Master Mix, 2X.
The purity and concentration of the extracted DNA sample (Number 9 and 10) was measured. The purity of the Sample 9 was A260/A280 2.11 and the concentration was measured as 137.36μG/μL. The purity of sample 10 was A260/A280 1.19, and its concentration was 149 μg/μl.
The purity of the extracted DNA of SFM have a direct effect on the amount of produced products, during the PCR reaction, and an adequate concentration is essential to have a sufficient amount to start the reaction.
Figure 1/ Designing the primers STR7:
Figure 2/ Gel of PCR products STR7 from SFM:
M 1 2 3 4 5 6 7 8 M
In Figure 2 the 2.2% agarose gel and PCR, reaction result in the gel is shown. On both right and left side of the gel the markers re visible which show the sizes ranges from 1500bp to 400pb. In this figure six columns, six different PCR products are available. The gel was used to find out if the primer did act in the PCR reactions or not. A mixture of 10uL of GO Taq is present in each well, consisting of 6.6uL of water, 1.2uL from the given primers, and 1uL of DNA temple. Since each well showed results and as the bands are present, we can assume that the reaction was successful. However, column number 8 is column containing the primer, which caused our concerns. This column consists of 2 bands which are considered to be heterozygous, while its top band is ~950bp and the bottom band is ~850bp. The estimated number of alleles is 8.
We extracted and purified the DNA of sailfin molly from a tissue sample in this project, which yielded in a sufficient amount of DNA. Thus, a high concentration 137.36μg/μl and purity of A260/A280 2.11 for sample 9 was achieved. This can be considered as a good concentration as it is not below 50 μg/μL, or even better to say a high concentration, due to the fact that the average concentration could be approximately 66μG/μL. Also as the purity is above A260/A280. 2, it can be considered as a good purity. In other steps, in order to design the primers, the DNA was blasted to locate the microsatellite region. With the help of the NCBI website for blasting of the primer, we were able to design the primer. In addition to blasting the primer, and in order to get a perfect primer we needed to modify the options. Initially the sequence contained 1-2000 nucleotides around the STR sequence. So we decided to change the TM to 60OC, the database allowed all organisms and we chose Poecilia latipinna reticulata as the organism window.
In order to have enough PCR products in PCR reactions, the samples were run through almost 30 time. The reaction can be considered successful as the PCR products were run through the agarose gel and eight PCR products were shown in the gel. The gel contained eight alleles, which contained five heterozygous samples. The heterozygosity in that sample was approximately [(5/8) x100] 62.5% of the total samples. During the determination of the relationship of the relatives (For example father and son), this would be an excellent method, as it can be used for both parental and forensic testing, due to the fact that each individual has his unique sequence. Only it must be taken into consideration that the reaction must be polymorphic to perform this method for both forensic and parental testing.