10
THE DEVELOPMENT AND ANALYSIS OF MICROSATELLITE DNA
Summary
Due to the significant negative impact of the industrial activities on the habitat, many species have become endangered. To conserve the species, a thorough investigation is needed. In this case, the research is based on the study the interdependencies between the genotype and spatial dispersal of populations.
Despite being endangered, turtles comprise rather specific object for such investigations, as well as any other long-lived organisms, because their reproduction cycles complicate the genetic investigations using conventional methods. That is why the novel DNA markers, microsatellites, were chosen for this research.
Although the microsatellites are very informative and universal, the overall cost for research is higher than for RAPD and AFLP. This is mostly due to the cost of the fluorescent dye which is used in the PCR. To eliminate this, I will apply an interesting technique which allows for incorporation of the dye by the forward primer into the product of polymerization thus saving a considerable amount of the dye.
Generally, I plan to base research on such steps:
- Studying the populations of Apalone spinifera and Apalone mutica based on available data;
- Taking samples of blood from the animals which are going to be caught by hand in their usual habitat. The sampling will occur in the least possible harmful way;
- Testing 12 pairs of primers initially developed for Apalone spinifera using the
20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.
(PCR), which isolates small fragments of DNA that have a high degree of variability from
Each human being has something called DNA. DNA is described as genetics and an extremely long macromolecule that is the main component of chromosomes and is the material that transfers genetic characteristics in all life forms. DNA constructs of two nucleotide strands coiled around each other in a ladder like arrangement with the sidepieces composed of alternating phosphate and deoxyribose units and the rungs composed of the purine and pyrimidine bases adenine, guanine, cytosine, and thymine. Each chromosome consist of one continuous thread-like molecule of DNA coiled tightly around proteins and contains a portion of the 6,400,000,000 basepairs that make up your DNA.
A. Rus hoelze et al, studied the impact of intense hunting of Northern Elephant Seals in the late 19th century, whose genetic variation reduced due to this bottleneck. This reduced their population size to just about 20. They found that although it has since rebounded to over 30K now but compared to their southern counterparts i.e., Southern Elephant Seals they have much less genetic variation. The Southern Elephant Seals didn’t went through this bottleneck event. It is clear that the genes of Northern Elephant Seals still carrying the marks of this bottleneck.
Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.
Flow Diagram Homework Assignment Name: Preetam Ganti Using the table below, explain the purpose of each of the steps of the Experimental Flow Diagram (Figure 1 from your lab manual – it is provided on the next page) for answering the two research questions guiding this laboratory investigation: 1. Is the bacterial contamination at these three farms due to the same plasmid or different plasmids? 2. What is the frequency of tetracycline resistant bacteria in the beef farm cultures?
Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).
The results show that under selection factors and environmental differences natural selection determines which allele should become more common. In the control simulation the frequency of white alleles to brown alleles, once this mutation was added, was about the same amount. It was almost half white and half brown. In simulation two the environment was an equatorial climate such as a forest and wolves were used as the predatory influence. Once the predatory factor was introduced it can be seen that the alleles of white fur decreased and at the end of the simulation the allele was almost lost. Thus, brown fur alleles were naturally selected in the equatorial environment. The fur color blends in with the environment helping them become harder to find by predators. Whereas, for the white bunnies their phenotype stood out in an equatorial environment causing them to be caught easily. Hence, it can be said that the brown fur alleles had a higher fitness which is why their occurrence was greater and that the white allele was less fit leading to less offspring being produced. Consequently, this supports my hypothesis that the brown fur allele would have a higher frequency in the equatorial environment.
Collecting samples on animals is very important,
Additionally, I. longirostris was genetically different among and across rivers. This first insights about the population genetic structure of I. longirostris is crucial for monitoring the genetic diversity, management and conservation of its populations and complementing the genetic studies in Prochilodontidae.
Variation in allele frequency through time will be the net result of colonizing and extinction processes in the population. In other words, while species abundance in the population may remain stable over time, genetic diversity may vary (Figure 1). For instance, we would expect populations at earlier stages of establishment to exhibit greater genetic turnover rate compared to populations that are well established (Figure 1). For the black-legged tick in southern Quebec, all populations regardless of establishment stage will likely be experiencing regular influxes of new genetic material from the introduction of new individuals. However, the relative amount of novel genetic material relative to that already present locally will differ depending on the establishment stage (Figure 1). For populations still undergoing recolonization-extinction cycles, the relative amounts of novel genetic material will likely be much greater compared to local genetic material due to high local mortality rates (Figure 1). In this case, we would expect to observe great amounts of genetic turnover over time, assuming that introduced individuals originated from multiple random source populations. Conversely, populations that are well established with a self-sustaining pool of local individuals would likely have enough local genetic material to mask the signal of any introduced material (Figure 1). Therefore,
In addition to the founder effect, loss of habitat and the potential for gene flow may have resulted in the discrepancy between the observed heterozygosity in Area
Genetic drift is often found in populations living on the islands because of the habitat isolation, the process of natural selection, interspecific and intraspecific competitions. Therefore, the species on the island always face the the limitation of food source, living space and predation issues, that’s why their population size is forced to decrease and sometimes extinct.
Captive breeding and reintroduction, translocations, population size estimates, inbreeding depression and avoidance, disease resistance, hybridization between introduced and native species, climate change and adaptation.
Different graph and statistics about the situation of endangered species in the world have been collected: