The aim of this experiment was to provide a good understanding of each step involved in PCR, provide possible patterns related to PCR and the changes in temperature, good skills on performing PCR and to provide good theoretical knowledge about Hardy-Weinberg model and skills on how to compare the real population`s genetic structure with the one found in Hardy-Weinberg `s equilibrium.
Transposable elements (jumping genes) are sequences of DNA that change their position on the genome and are found in most of the living species. This phenomenon occurring in the genome was discovered by Barbara McClintock . The 2 major divisions of transposable elements are retrotransposons and transposons. Retrotransposons are found in the first type of transposable elements and are characterized by the ability to use reverse transposase to make RNA intermediate needed for transposition. They are usually positioned in the genomes of the viruses and some of the most known examples are LINES and SINES. Integrase and reverse transcriptase needed for transposition are encoded by retrotransposons. They proceed in a copy-paste mode .
The transposable elements of the second type are transposons, which proceed by a cut-paste mode and they usually carry other genes. Transposase is used to support this mode of proceed and it can bind either to a specific DNA sequence or to the end of the transposons that contain inverted repeats. Some of the most known examples of this type are corn Ds element and fruit fly element .
Transposase (Tn5) assists in the cut-paste mode of proceeding by binding to transposons, characterized by a donor DNA that leaves away from its original site toward a new site in the genome .
Alu elements are found in genomes of primates. They are short pieces of DNA influenced by the action of Arthrobacter luteus restriction endonuclease. They are retrotransposons, resembling to the RNA` encoded RNA polymerase III. In humans it is found in the form of SINE, approximately 11 % of human`s genome and the most repeated form is PV92, that is found on the 16th chromosome. They are characterized by a base length of 300 bp .
PV92 exhibits polymorphism, so it can either be or not on the 16th chromosome and 3 different phenotypes are expressed respectively: PV+/PV+, PV+/PV-, PV/PV-, where phenotype is not related to either recessivness or dominance. Most of the studies related to it include DNA isolation from a specific region of human body, ex. the mouth; amplification of PV92 region by usage of Polymerization Chain Reaction and Gel electrophoresis that is crucial in determining the polymorphism`s length and whether Alu insertion is present or not .
Alu insertions are transmitted from ancestors to the offspring. Most of them are positioned in the non-coding regions, and show evolutionary neutrality. In neurofibromatosis I, an Alu element inserts in NF-1 gene that causes malfunction in tissue plasminogen activator (TPA) and the angiotensin converter enzyme (ACE) and displays problems in heart .
Alu mutations have a fixed mode and the 2 chromosomes of same pair develop the insert at the same locus . Due to the phenomenon of dimorphism difference in allele and the frequencies of the genotype arise, evolutionary studies are able to be performed .
Polymerase chain reaction is used for the amplification of DNA samples and is characterized by 3 important steps, which are: denaturation, annealing and extension and in each step the DNA doubles. In order that this process proceeds, some specific elements are needed, such as: MgCl2 which induces the production and supports the activity of Taq DNA polymerase; DNA polymerase needed for the reproduction of new strands of DNA; dNTP or the monomers of DNA and concentrated (10X) buffers .
Hardy-Weinberg Law denotes the maintenance of the genetic variation in a specific environment, when the factors, such as: genetics drift, gene flow, nonrandom mating, natural selection and mutations are not present .
MATERIALS & METHODS
Firstly 10 ml saline sample of a 0.9% concentration (same as the blood`s concentration) was taken inside the mouth from each student respectively and kept for 30 seconds. After that the saline solution is taken out from the mouth by swirling and is positioned in the same sample where it which was firstly positioned. A new 1.5 ml was taken reciprocally by each student and labeled with the reciprocal initials and 1 ml of the solution was placed inside it by the usage of a micropipette. Each of the samples is centrifuged in a microcentrifuge for one minute. The pellet positioned in the bottom part of the tube was kept while the other part was thrown away. In the cases where the pellet was not in an appropriate amount saline was placed again in the tube and afterwards the tube was centrifuged. Chelex (resin containing organic molecules that enable the taking off of the metals from DNA during boiling) tubes of 100 microliters (10%) were taken and labeled with the reciprocal initials. 1 ml from the cell suspension is placed inside the Chelex. Each of the tubes were heated in the thermo-cycler and later centrifuged for one minute. Other tubes were taken, one for each reciprocally and labeled. 100 microliters from the Chelex tube was positioned in the new tube. Later a compound of PCR is prepared by using 2.5 ul 10X PCR buffer, 1.5 microliter of MgCl2, 0.5 ul dNTPs, 0.5 ul PV92 F primer, 0.5 ul PV92 R primer, 0.25 ul Taq DNA polymerase and 16.75 ul sdH2O. 2.5 ul of DNA samples reciprocally are positioned in the tubes of PCR. After being mixed and centrifuged, the samples were put on ice. The process of amplification is run in the conditions of 30 cycles, specifically 94OC/30 seconds; 68OC/30 seconds and 72OC/30 seconds. After a week, the third sub-part of the experiment was performed, the gel electrophoresis. Firstly the buffer inside the graduated cylinder was shaken in order for its concentration to be maintained. Both 2 sides are cleaned and the appropriate well is banded. A gel of volume of 150 ml was prepared by using 1.5 ml agarose that was firstly measured on a scale. This gel was put inside an Erlenamyer flask. 1X TAE was placed inside the graduated cylinder. The proper well is used to be tempered around the two sides, in order to prevent the band from staying there glued. The mixture is boiled in a microwave and after the boiling 6 microliters of Ethidium bromide is added, in order to ensure the release of DNA due to the ability of it to enter in the bases of DNA. After the mixture starts to give off the heat and the steam level decreases, it is placed toward the middle on the appropriate well without creating any bubbles. Afterwards, a period of time of 37-45 minutes was required about the gel to freeze. Later the buffer was loaded respectively by each group and our samples were loaded too. Our group`s samples were positioned in the 2nd row 5-13. 1 kb ladder was loaded in the left part of the second row in the 14th position. The electrophoresis at 130 V for 30 minutes followed after those steps and lastly the gel photograph was taken and the data were recorded.
|Dominant character||Recessive character|
|No. of people with this trait||7||23|
Table1: The number of people showing or not the bent little finger phenotype, in a specific environment (in the class in our case)
P2 + 2PQ + Q2= 1
P + Q =1
P- the frequency of the dominant allele in a population
Q- the frequency of the recessive allele in a population
Q2– frequency of the recessive phenotype of the character in a population = 23/30 = 0.767
P=1-Q= 1- 0.029 = 0.971
p2 + 2pq = 1-q2= 1-0.767= 0.233
The values of dominancy are not the same, as it is shown that the observed frequency of dominance in our population is 0.971, but the dominance according to our calculations is approximately 0.233, so our population is not in equilibrium according to hardy-Weinberg population.
The reason why the results of gel electrophoresis were no able to show the results we were expecting, with the shortest DNA pairs going faster and the longest ones standing in the behind font can be because of the small amount of DNA absorbed from the mouth or the non-purities existing in this section that affect the DNA`s magnitude.. Another possibility can be that the primer was unable to attach DNA and made the PCR unable to be realized, Also range of temperatures can have affected a failure in PCR. On the other hand, the probable reason why the class`s population is not in Hardy-Weinberg population is because of the rareness that the populations in such equilibrium are found. Most of the populations are in a finite number, supporting in this way a violation to this equilibrium, also affected by the natural selection. Also as the system of study we have chose is not an isolated one, the violation to the equilibrium is supported too. Although the trait we studied does not affect the mating between 2 persons as would other traits affect, such as height.
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