Haemochromatosis Detection Essay

For millions of years, repetitive DNA sequences such as the “Alu sequence” have been randomly inserted in the human genome. In this experiment, I will study whether or not the repetitive element such as the “Alu sequence” is present in the PV92 region of chromosome 16 of my DNA. I will then gather the data from my Biology Lab Class and compare it with those of the United States. To perform this experiment, the method of Polymerase Chain Reaction (PCR) will be used to amplify the DNA. The purpose of using PCR is to create enough mass in order for it to run on the agarose gel electrophoresis. Performing this

Hemochromatosis is hereditary and the most common genetic disease in the U.S. This genetic disease is passed on

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

The following results helped obtain the haplogroup that in which the sequence of mtDNA would identify. The PCR reaction worked, and this can be determined by looking at the agarose gel in figure 1. If the PCR reaction was successful, than a band should appear around 550bp. Individual AC displays a band around 550bp, this means the PCR reaction was successful. The band for individual AC, depicts a low concentration of product, because the band faint. After the purification process the concentration, A260/280 ratio, and A260/A230 ratio were determined by using the nanodrop. The concentration of mtDNA in the product was 60.9 ng/uL. The ratio for A260/280 was 1.79 and the ratio for A260/230 was 0.77. The A260 and 280 are a spectrometer measurement that measure absorbance at wavelengths of

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

The predicted antigenic regions were chimerically expressed in a bacterial system. Briefly, two sets of PCR primers were designed according to gD gene sequence available in GenBank (accession number: NC_001847) to amplify gene fragments encoding aa 20-160 (nucleotide 118953～119375, Δ gD1) and aa 257-344 (nucleotide 119664～119927, Δ gD2), respectively. The primers sequences for amplification of Δ gD1(P1 and P2) and Δ gD2 (P3 and P4) were listed in Table. 1.

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).

I hypothesize that there will be a strong deviation between the genotype frequencies of the two lab sections combined and that of the US. This experiment consisted of 26 individuals, of varying ethnical backgrounds, ages, and genders, who amplified their own DNA through the use of PCR. Their DNA was examined for the presence [(+/+) or (+/-)] or absence [(-/-)] of the Alu insertion through the use of gel electrophoresis.

The gel was covered with a buffer and then six samples labeled A-F were deposited into the wells using a micropipette. Three of these samples (A, B, C) were control samples to compare to D, E, and F (the mother, child, and father). The safety cover was placed on the unit and then brought to a power source. The leads were connected the chamber and left for approximately 20 minutes. The agarose gel was removed from the tray and placed onto a sheet of plastic wrap. An Ethidium Bromide card was placed face down onto the gel to stain for approximately 5 minutes. Finally, the card was removed and the gel was placed on top of a UV light. The samples were pushed towards the center due to opposite electric charges. Agarose gel separates the DNA samples by the way they were cut. The restriction enzyme MST II cuts the DNA strand at CC/TNAGG where N is any nucleotide base. If the enzyme recognizes this, it is cut. If it does not recognize it then the strand is left whole. We were able to observe the DNA strands due to them being dyed and placed over a UV light. The control samples were utilized so that the other samples could be compared to test for their genotype. The data was analyzed in this way to differentiate between the different genotypes and the number of bars they

Throughout analysis of the data, we complete DNA agarose gel electrophoresis in order to determine the size of our plasmid DNA. The electrophoresis

In 1990s, several molecular studies on RH genes (20-22), confirmed that there are only two genes (RHD, RHCE) encode Rh proteins. RHCE gene was discovered and cloned in 1990, two years later the RHD gene was cloned, these genes reside in close proximity on the short arm of chromosome one, and inherited together as codominant alleles. RHD gene encodes RhD protein, while RHCE gene encodes the Cc and Ee antigens in various combinations (Rhce, RhCe, RhcE, or RhCE).

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

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This essay based on the principle of real time PCR which uses CYBR green dye that combines to any double stand DNA. This process included two maim steps. The first step was designing of HSV-1 primer and /or HSV-2 primer (we have chosen only HSV-1). BioEdit software has been used to edit the nuclide sequences where necessary. After that the primer has been ordered. The second step was in the laboratory which included applying the HSV-1 primers to real time PCR protocol. The final results of this protocols aim to demonstrate the quality of HSV-1 primer that we have designed by interpretation three criteria. These are specificity, efficiency and