|✅ Paper Type: Free Essay||✅ Subject: Sciences|
|✅ Wordcount: 1690 words||✅ Published: 1st Jan 2015|
For centennials scientists and laymen alike have been awe-inspired and intrigued by the phenomena that is Alu elements. It is a family of short interspersed repeats that have mobilized throughout primate genomes by retrotransposition over the past 65 million years of primate evolution (Comas et al., 2001).
Alu elements are regarded as Sort Interspersed Nuclear Elements (SINES) whose length spans 300bp. In close proximity to the centre of the Alu element itself, is a recognition site for the restriction enzyme Alu I, of which its name is ascertained. These Alu elements accounts for 5% of the human genome and is believed to have arisen from a gene that encodes the RNA component of the recognition molecule (Smith, 2005).
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Alu is a transposable DNA sequence that has the ability to repeatedly copy and insert itself into new chromosome loci i.e. “jumping-genes”. More specifically Alu is a retroposon – it necessitates the retrovirus enzyme reverse transcriptase to produce an identical copy of itself that has the capacity to be mobile. This is achieved when the inserted Alu, by processes of RNA polymerases, is transcribed into mRNA. The newly formed mRNA is converted to a double stranded molecule. This is accomplished by the action of reverse transcriptase. In the end, at any new chromosomal locus at the site of a single or double stranded break, the DNA copy of Alu is integrated (Robinson, 2005).
In order for an Alu element to self transcribe, each has an internal promoter for RNA polymerase III. Nevertheless, it lacks the ability to produce a copy of itself. Furthermore, to integrate this copy into a new chromosome position. But other transposons the very functions Alu lacks. One of these other transposons is L1, a Long Interspersed Nuclear Element (LINES), as a results of LINES ability to retain a functional reverse-transcriptase gene. In addition to LINES ability to reverse transcribe RNA to DNA , L1 reverse transcriptase is also able to produce single stranded nicks in DNA. If a chromosomal locus contains the sequence AATTT, the reverse transcriptase enzyme will produce a nick the polyadenalated tail of the Alu transcript hydrogen bonds to the TTT sequence at the nicked site. Thus creating a primer for for the reverse transcription. Enabling the DNA copy to migrate (Fisher, 2007).
This representation depicts the efficiency of L1. Moreover, how L1 provieds the the fuctions for Alu tranposons. In essence, it shows how Alu is a parasite of L1 i.e. a remnant of an ancestoral retrovirus (Kass et al., 2007).
This study examines PV92, a human-specific Alu insertion on chromosome 16 and which belong to the subfamily Alu Y. The PV92 genetic system has only two alleles indicating the presence (+) or absence (-) of the Alu transposable element on each of the paired chromosomes. It is aimed at extracting DNA, amplifying it and running it on a 2% agrose gel. The results attained will be analysed for the possible Alu insertion or deletion. It is hypothesised that with the aid of specific molecular biology techniques, analysis of the PV 92 Alu element can be achieved and the population will be in Hardy-Weinberg Equilibrium.
Method And Materials
A) DNA Samples
For approximately 1minute, sterile nylon swabs were rubbed inside the cheek. The swabs were then placed in 250µl of QuickExtract and rotated in such a manner that no solution was spilt. Thereafter, swabs were pressed against the sides of the tubes, removed the tubes were closed. The tubes were vortexed for 10seconds and incubated for 1minute at 65°C. A second vortex then followed for 1seconds. The tubes were then incubated for 4minutes at 98°C. Upon completion, the tubes were re-vortexed for 15seconds. With the use of a nanodrop, the DNA of each tube could be quantified. The expected yield for each tube was 20ng/µl.
B) PCR Conditions
In a final volume of 150µl, a standard master mix was prepared. Each master mix comprised 1x reaction buffer, 200mM dNTP mix, 1.5mM MgCl2, 1µM of each primer and 0.016U/µl of Taq polymerase. Following a 1:1 dilution, the DNA stock was diluted to a concentration of 15ng/µl. The reaction was set up by mixing 15ng of DNA to a master mix which was made up to a final volume of 25µl. Finally, the preparation of the negative control followed containing 24µl of master mix which was made up to a final volume of 25µl.
The reactions were then run for 30 cycles by means of the subsequent constraints: 95°C 1min, 55.5°C 1min, 72°C 1min and 72°C 7min. This was then held at 4°C.
C) Gel Electrophoresis
In an Erlenmeyer flask, 2g of agrose powder was weighed out and 100ml of 1x TBE was added. The agrose was dissolved by heating in a microwave oven. The solution was then cooled and 1µl of EtBr was added. The agrose was then poured into a casting tray with a comb and this was allowed to set at room temperature. Of the PCR product, 10µl was mixed with 1µl loading dye. Then 10µl of the newly formed sample was loaded onto the gel alongside the MW marker. Using a UV light, the DNA fragments could be visualised and photographed. The size of the amplified product could then be attained.
Frequencies for a class of 70 students
++ = 35 individuals
+- = 30 individuals
— = 5 individuals
Therefore the total number of alleles = 140
Allele frequency distribution of the ++ and — allele in the Normal Population
Allele frequency for ++ allele = [(++ x 2) + (+-)] ÷ (n), where n= number of alleles in the whole population
Therefore: Allele frequency for ++ allele = [(35 x 2) + 30] ÷ (140)
Allele frequency for — allele = [(– x 2) + (+-)] ÷ (n), where n= number of alleles in the whole population
Therefore: Allele frequency for L allele = [(5 x 2) + 30] ÷ (140)
Observed Frequencies (OF)
OF = Number of individuals for a specific allele ÷ total number of individuals in the sample
Therefore: ++ allele = 35 ÷ 70 = 0.50
+- allele = 30 ÷ 70 = 0.43
— allele = 5 ÷ 70 = 0.07
Hardy-Weinberg Equilibrium (HWE)
The formula for determining HWE = p2+2pq+q2
Therefore: HWE = (0.714)2 + [2(0.714 + 0.286)] + (0.286)2
Comparison of observed frequencies with expected frequencies
Observed Expected (O-E)2 ÷ E
++ 50 51 0.0196
+- 43 41 0.0976
— 7 8 0.1250
Total ∑ 0.242
For degrees of freedom = 1 and a 95% probability, the critical value attained from the Chi square table is 3.843
The results attained depicted that 35 individuals in the sample were homozygous for the insertion, while 30 were homozygous for no insertion. Only 5 individuals in the sample were heterozygous. This shows that a greater percentage of the population can pass on the PV92 insertion to their offspring.
The allele distribution frequencies of the ++ and +- allele in the normal population revealed that 71.4% of the population was ++ which is just below 2½ times more that the 28.6% attained for the – allele.
The observed frequencies materialised similar results. The ++ allele had the highest frequency with +- having the lowest. Once more, the – allele was the intermediate.
When comparing the observed frequencies to that of the expected frequencies, the critical value attained was 0.242. With the respects to the Chi square table, for degrees of freedom being 1 and for a 95% probability, the critical value was 3.843.
The Hardy-Weinberg principle implies that both allele and genotype frequencies in a population remain stable i.e. equilibrium, from cohort to the next except if specific disturbing influences are launched. Those comprise non-random mating, mutations, selection, limited population size, “overlapping generations”, random genetic drift and gene flow (Wikipedia, 2010 and Roux, 1974).
With the above mentioned, it was attained that the sample was found to be in equilibrium as the calculated critical value was less than the one attained from the Chi square table at a 95% probability.
Therefore, it can be said with much certainty that the hypothesis made was true.
* Comas, D., Plaza, S., Calafell, F., Sanjantila, A. and Bertranpetit, J. (2001). ‘Recent Insertion of an Alu Element Within a Polymorphic Human-Specific Alu Insertion’ European Journal of Pharmacology – Molecular Pharmacology Section, 247, 239-248
* Fisher, L. (2007). Alu Frame-set. (Online) (Cited 28 March 2010) Available from http://www.geneticorigins.org/pv92/aluframeset.htm
* Kass, D., Jamison, N., Mayberry, M. and Tecle, E. (2007). ‘Identificatin of a unique based Alu-polymorophism and its use in Human Population studies’. Journal of Genes.
* Robinson, N. (2005). PV92 Locus Alu: How to Track Human Migration Following This Gene Insertion. (Online) (Cited 28 March 2010) Available from http://www.associatedcontent.com/article/1452910/pv92_locus_alu_how_to_track_human_migration_pg2.html?cat=58
* Roux, C. (1974) Hardy-Weinberg Equilibria in random mating populations. Theoretical population biology. 5: 393- 416
* Smith, A. (2005). What is PV92? (Online) (Cited 28 March 2010) Available from http://www.fbr.org/swksweb/pv92.html
* Wkipedia (2010). Hardy-Weinberg principle (Online) (Cited 28 March 2010) Available from http://en.wikipedia.org/wiki/Hardy%E2%80%93Weinberg_principle
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