Hereditary Haemochromatosis Lab Report

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).

PCR genotyping utilizes restriction fragment length polymorphism (RFLP). This method uses restriction enzymes to cut the DNA at specific sequences. Then, these segments of DNA are amplified through three steps: denaturation, annealing, and elongation. Denaturation breaks the double stand. DNA primers anneal to the DNA template during annealing. During elongation, the DNA primers add nucleotide bases to complement the template strand. Then, place the PCR products int the electrophoresis gel to separate the DNA by size (smaller will move faster, and bigger will move slower). This is the crucial step that will show one if the PCR produced the desired DNA fragment that one is genotyping. The DNA ladder contains the DNA fragments of known size which will be used to compare to the bands produced in the other lanes (the PCR products). If the PCR product contains the DNA fragment, then the PCR bands and the DNA ladder bands should match up. My gel electrophoresis sample did not show due to incorrect techniques. My samples might not have been loaded into the wells correctly. However, one should expect to see the wild-type fly at 467 bp, white fly at 704 bp, and the unknown in lane 3 both the 467 and 704 bps. Female flies are homozygous recessive. As seen in all of the white crosses, female white flies are very rare compared in t number to the male white flies. Therefore, one can infer that female offsprings must inherited 2 alleles for the white eye color to show in females. The WT male x w female cross shows that female white flies inherit the white eye phenotype in a homozygous recessive

The mineral iron (Fe) is a crucial substance for the human body. Many parts of the body, including red blood cells, require iron in order for the body to function normally. Only specific amounts of iron are absorbed by the body to maintain homeostasis. However, iron is a very toxic mineral so if there is too much iron stored in the body, it can lead to serious consequences. People that suffer from haemochromatosis, a hereditary iron disorder, are unable to regulate the amount of iron absorbed from their diet. Almost all iron in the intestine is absorbed into the blood stream and because the human body has no major way to rid of excess iron, it is stored in organs and joints of the body where it can cause serious harm. Typically, the body stores one gram or less of iron but people with haemochromatosis can have up

Hemochromatosis can be treated through a process called bloodletting. It is a practice where a patient is purposely bled. Regularly bleeding a hemochromatosis patient can reduce their iron to normal amounts, which prevents severe damage to the organs. Therefore, although hemochromatosis leads to death in a few decades, “it will protect us from a disease that is killing everyone else long before

Hemochromatosis is a genetic disorder where you body absorbs excess iron from the food that you eat. Your body then stores the excess iron in your crucial organs like your heart, brian, pancreas, and liver. Your body also stores the iron in your joints. If too much iron were to accumulate in any of your vital organs it could lead to organ failure and even

The Blue sample, which was hypothesized to be recombinant, should have produced similar if not identical bands to the known recombinant Green sample, which is not the case in the PCR gel. Instead, the Blue sample shows faint bands more closely resembling the Red sample, known to be non-recombinant. The bands did not show through for the PCR samples which indicates that the PCR preparation was not successful. This is further confirmed when analyzing the positive control which produced no results. The lack of bands in the negative control suggest that failure this portion of the experiment was not due to

Specific methodologies are implemented in DNA fingerprinting to get a reliable and valid result. The different steps are – isolation, cutting, sorting, analysing of DNA.

In order to justify some of the benefits of the hemochromatosis gene, it is important to mention that a hemochromatic person has an uneven concentration of iron in their bloodstream. Although there is plenty of iron to go around, not all of it is distributed evenly. Certain white blood cells called macrophages are among those cells in the bloodstream that do not receive a sufficient amount of iron in a hemochromatic bloodstream. Normally, macrophages act as guards for the blood against foreign invaders, and when they find a virus, they bind to it and bring it back to the lymph nodes for filtering the foreign particle. Sometimes, this process backfires. Since (almost) all organisms on Earth thrive on iron, the virus that is attached to the macrophage starts to feed on the iron in the cell. By the time the macrophage and virus reach the lymph node, the virus is stronger after consuming the iron, and is able to multiply and invade the lymphatic system. In a hemochromatic person, however, the macrophages are starved of

We then added a wash solution to further purify the sample and centrifuged the solution again removing the eluate once more. We then centrifuged the tube for an additional 2 minutes and emptied the eluate. We then transferred the spin tube to a clean PCR tube. Which we then added 30 ul elution solution and incubated for 1 minute at room temperature and then handed off the sample to our TA to be centrifuged at a higher setting for 1 minute. We then discarded the spin column and prepared another PCR tube with 6 uL sterile water, 2uL DNA primer, and 2 uL of clean PCR product. We then handed off the tube to the TA to be processed by the sequencing

Hereditary Hemochromatosis is a disease that causes the body to absorb and store more iron than is needed. Iron is an important nutrient that people get from food. It is the main component of hemoglobin, and it carries oxygen away from the lungs to the rest of the body. In Hereditary Hemochromatosis, the body absorbs almost twice as much iron as it should be, that causes it to be stored in other areas such as joints and major organs. This excess iron cannot leave these joints or major organs, Over time this can cause diabetes and damage to an organ. Even though a kid could be born with hemochromatosis, the disease might not be recognized or diagnosed till early adulthood or later. This is caused by a virus in a gene that controls how much iron the body takes in from one’s meal. This genetic disease alters the regulation of iron absorption. If correctly diagnosed, HH is easily and effectively treated, but if untreated, it can lead to severe organ damage.

Hereditary Haemochromatosis (HH) is an autosomal recessive heritable disease characterized by abnormally increased iron absorption from the small intestine, causing iron deposition (Jansen and Swinkels, 2009). Excess iron is deposited in the liver, heart, joints, pancreas and other endocrine organs (e.g. adrenal glands), resulting in disruption in the organs’ function (Limdi and Carmpton, 2004). Damages in tissue of the organs mentioned, can possibly cause inter alia, (table 1) cirrhosis, diabetes, heart failure or even death (McCance et al, 2014; van Bokhoven et al, 2011).

Genes are small segments of DNA (on a specific locus of a chromosome) that contain the code used to synthesise a protein and mRNA molecule (Khan Academy, 2014). The ‘normal’ function of the HFE protein involved with haemochromatosis is to regulate the production of the protein hepcidin, produced in the liver, which determines levels of dietary iron absorption and the it’s release from storage sites in the body (Haemochromatosis.org.au, 2014). The human body limits iron stores through the HFE protein, however hereditary haemochromatosis is caused by a mutation of the HFE protein in the DNA, means it cannot communicate with the transferrin receptor. As a result, when iron enters the body, the transferrin absorbs the mineral into the cells.

These molecular-based techniques have been revolutionised by the discovery of endonuclease enzymes and the invention of the Polymerase Chain Reaction (PCR) in 1983 by Kary Mullis. This technique allows for the rapid cloning of fragments of DNA so that further testing can be carried out and has simplified the process of nucleic acid amplification in vitro (Bartlett and Stirling, 2003). Using a

PCR tubes were ready for purification when a single DNA band was present per lane on the agarose gel. If multiple bands were obtained in a single lane the PCR process and gel visualization process was repeated however, the denaturation temperature was run at a higher temperature to prevent non-specific binding of the primers to the DNA template. The purification process used an Invitrogen PCR clean-up kit. 5mL of charge switch beads

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.