Hereditary Haemochromatosis Lab Report

RAPD is effective in screening the differences in DNA sequences. The RAPD markers are also DNA fragments from PCR amplification,

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).

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

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.

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

DNA is isolated from the blood sample and amplified through the polymerase chain reaction (PCR), a technique for rapidly producing,

The enzymatic or the molecular “xeroxing” process in which a particular region of DNA is replicated several times to yield thousands of copies of a specific sequence is termed as Polymerase chain reaction (PCR). The process of PCR employs a sophisticated and precise pattern of heating and cooling of samples in a thermal cycler, over 30 cycles.

Hereditary spherocytosis (HS) is a type of anemia that can be passed along from parent to child (inherited). Anemia is the condition of not having enough healthy red blood cells in the body. Red blood cells are made in spongy tissue inside of bones (bone marrow) and they carry oxygen in the blood.

The IL-10 promoter polymorphism (PCR-Restriction Fragment Length Polymorphism) assay as described by Turneret al(year) is used for detecting the -592 SNP by introducing 2 alleles, A and C (A-C). A measurement of 100 ng of genomic DNA is amplified in a 25 mL and final volume reaction mixture containing 10X KCL buffer, 1.5mM MgCL2, 0.2mM of each dNTP, 25 pmol of each primer, and 1 unit of Taq polymerase was determined. The cycling conditions were determined as follow: first, in 95°C for 5 minutes; and second 35 cycles of 94°C for 40 seconds at 61°C for 1 minand at 72°C for 40 seconds were specified; finally at 72°C for 10 minutes it was determined. The PCR product contains 412 bp and the Restriction Fragment Length Polymorphism assay was performed in a 15 mL reaction mixture containing PCR product (10 mL), buffer (1.8 mL), enzyme (RsaӀ 2 units per reaction), and distilled water (3 mL). The reaction

Random amplified polymorphic DNA (RAPD) markers are analyzed by using PCR to amplify the segments of nuclear DNA. The use of a single primer (usually 8–10 bp long) that attaches to both strands of DNA and low annealing temperatures increases the likeli¬hood of amplifying multiple regions representing a particular locus (Welsh and McClelland, 1990). Although RAPD is a simple and inexpensive technique its major limitation is the inability to differentiate between homozygote and heterozygote; this marker is therefore regarded as a dominant type. This technique is based on the use of short, arbitrary primers in PCR reaction and can be used to produce relatively detailed and complex DNA profiles

Reverse transcriptase polymerase chain reaction, also known as RT-PCR, has been recognized as a reliable, accurate, and sensitive method for quantifying gene transcription. Polymerase chain reaction, also known as PCR, is considered an essential tool in molecular biology that allows for the amplification of nucleic acid sequences. Specifically, the three main consecutively repeating steps in PCR are denaturation, annealing, and elongation. If the reaction runs with 100% efficiency, there will be a two-fold increase in target amplicons after each cycle of PCR. Therefore, with n cycles of the reaction, the copy number of the target sequences will be 2n. However, one of the main disadvantages of conventional PCR, also called end-point PCR, is that the results of amplification can only be visualized after all the cycles of amplification are complete (Nestorov 326).

PCR reaction was carried out in a 25 μl reaction mixture under standard condition, contained 1x buffer, 0.2 mM of four dNTPS, 1.5mM mgcl2, 1 pM of forward (sequence) and reverse (sequence) primers and 50ng of genomic DNA. Initial denaturation at 95˚C for 4 min was followed by denaturing at 94˚C for 30 seconds, annealing at 60˚C for 30 seconds and extension at 72˚C for 30 seconds for 32 cycles. The final extension was 8 min. The amplicons were run on an ABI 3130x (Applied Biosystems, Foster City, California,

PCR reaction is typically performed in a thermocycler also called as a PCR machine or DNA amplifier.

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.

The polymerase chain reaction in molecular biology also allows scientist and medical professionals to replicate copies of specific DNA sequences in millions in a matter of few hours. In plain language, this allows researchers to photocopy or Xerox specific DNA sequences in a short period of time. They to replicating or duplicating specific DNA sequences however is a familiarity of a part of the sequence of the DNA molecules. This primers can be later synthesized to