Alu elements

Alu ingredientsIntroductionFor 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 one(a)time(prenominal) 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).Alu is a transpos suitable DNA sequence that has the ability to repeatedly replica 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 monovular 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 desolate 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. Further much, 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 tra nscriptase 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 undersurface 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 movement (+) 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 analys ed 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 chamberpot be achieved and the state will be in Hardy-Weinberg Equilibrium.Method And MaterialsA) DNA SamplesFor approximately 1minute, sterile nylon swabs were rubbed inside the cheek. The swabs were then placed in 250l of QuickExtract and rotated in such a modal value 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 65C. A second vortex then followed for 1seconds. The tubes were then incubated for 4minutes at 98C. 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/l.B) PCR ConditionsIn a final volume of 150l, a ideal master mix was prepared. Each master mix comprise d 1x reaction buffer, 200mM dNTP mix, 1.5mM MgCl2, 1M of each primer and 0.016U/l of Taq polymerase. Following a 11 dilution, the DNA stock was diluted to a concentration of 15ng/l. The reaction was set up by mixing 15ng of DNA to a master mix which was do up to a final volume of 25l. Finally, the preparation of the negative control followed containing 24l of master mix which was made up to a final volume of 25l.The reactions were then run for 30 cycles by means of the subsequent constraints 95C 1min, 55.5C 1min, 72C 1min and 72C 7min. This was then held at 4C.C) Gel ElectrophoresisIn 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 atomise oven. The solution was then cooled and 1l 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, 10l was coalesce with 1l loading dye. Then 10l of the newly formed sample wa s 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.ResultsFrequencies for a class of 70 students++ = 35 individuals+- = 30 individuals = 5 individualsTherefore the resume arrive of alleles = 140Allele frequency distribution of the ++ and allele in the Normal PopulationAllele frequency for ++ allele = (++ x 2) + (+-) (n), where n= anatomy of alleles in the whole populationTherefore Allele frequency for ++ allele = (35 x 2) + 30 (140) =0.714 =71.4%Allele frequency for allele = ( x 2) + (+-) (n), where n= number of alleles in the whole populationTherefore Allele frequency for L allele = (5 x 2) + 30 (140) =0.286 =28.6%Observed Frequencies (OF)OF = Number of individuals for a specific allele total number of individuals in the sampleTherefore ++ allele = 35 70 = 0.50 +- allele = 30 70 = 0.43 allele = 5 70 = 0.07Hardy-Weinberg Equilibrium (HWE)The fo rmula for find HWE = p2+2pq+q2Therefore HWE = (0.714)2 + 2(0.714 + 0.286) + (0.286)2 = 1Comparison of observed frequencies with expected frequencies Observed Expected (O-E)2 E++ 50 51 0.0196+- 43 41 0.0976 7 8 0.1250 substance 0.242 For degrees of freedom = 1 and a 95% probability, the critical value attained from the chi square table is 3.843DiscussionThe 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 2 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, th e 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 cat 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.References* Comas, D., Plaza, S., Calafell, F., Sanjantila, A. and Bertranpetit, J. (2001). upstart 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 marching 2010) unattached 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