PCR Gel Electrophoresis
Introduction
Sickle cell anaemia is caused by a Single Nucleotide Polymorphism (SNP). SNPs are the most common type of genetic variation, with each SNP representing a difference in a nucleotide. SNPs occur normally throughout a person’s DNA. Most commonly, they are usually found in non-coding regions and have no effect on health. When a SNP occurs in a coding region, it may play a direct role in disease by affecting the gene’s function. This SNP for sickle cell anaemia produces the recognition sequence for a restriction enzyme, HindIII. Samples without the SNP (not carrying the disease) do not contain the recognition sequence, and are not cut. This allows the digested DNA fragments to be analysed by being separated with the use of gel electrophoresis, enabling the possibility of identifying whether a patient has sickle cell anaeminia.
Before we can visualise the DNA we need to amplify the DNA. In order to amplify the DNA we are going to use a technique called Polymerase chain reaction (PCR). With the invention of the PCR technique, DNA profiling took huge strides forward in both discriminating power and the ability to recover information from very small (or degraded) starting samples. PCR greatly amplifies the amounts of a specific region of DNA, using primers and DNA polymerase. The PCR method is readily adaptable for analysing STR loci.
The PCR method relies on cycles of repeatED heating and cooling of the reaction for the denaturing of DNA and enzymatic replication of the DNA primers containing complimentary DNA to the target region, with the DNA polymerase synthesising the reaction. The DNA polymerase used is normally Taq polymerase as it is very thermostable. As the PCR progresses, the DNA generated ...
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...ow any bands, which shows there has not been any contamination. These results imply that the experiment has been a success as no problems were found.
All five samples were used to increase the reliability of the experiment. The PCR without DNA is used as a negative control, no band was expected which ensures that there is no effect when there should be no effect. The PCRs with DNA which are not cleaved are to used compare the cut and uncut DNA. Also, it ensures that there is an effect when there should be an effect.
An improvement to the experiment could be including a digest containing fragments of known sizes to be used as a guide to confirm that the cleaved DNA is of the expected size
References http://peds.oxfordjournals.org/content/13/4/283.full http://ghr.nlm.nih.gov/handbook/genomicresearch/snp
http://www.ndsu.edu/pubweb/~mcclean/plsc431/students/firas.htm
When the PCR technique is completed, the tubes are stored at 4°C until analysis of the tubes. To analyze the PCR results with the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel for the electrophorese consists of 1.5% agarose gel with 0.5x TBE and 200ng/ml ethidium. bromide. The sand is a sand.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
Having an inherited disease like sickle cell anemia takes a life-long of treatments and preventions of complications that comes with being diagnosed with sickle cell anemia. Many people have lived a long time while dealing with the disease because of the on-going care being provided to keep patients as healthy as possible. Knowing that both male and female have the sickle cell trait can prevent having a child with the sickle cell disease. From the help of the parents, doctors and medicine, sickle cell patients don’t have to deal with the pressure of living with a life threatening disease.
DNA fingerprinting, or sometimes known as DNA typing, is isolating and developing images of sequences of DNA to evaluate the DNA in an individual’s cells. DNA fingerprinting today is used for many different things in many different areas of science. In forensic science, DNA typing can determine which person did which crime by using blood or skin left at a crime scene. In medical science, patients can find out who their siblings, parents, or children are by using DNA fingerprinting (webmd).
Hemoglobin SS happens because of a mutation in chromosome 11. Chromosome 11 contains the gene of hemoglobin-Beta. Hemoglobin (HBB) transports oxygen to your body parts like your lungs. Hemoglobin contains 2 alpha hemoglobin and 2 beta hemoglobin chains. Sickle cell anemia results from a point mutation. There is a change in the sixth amino acid in the beta hemoglobin chain from GLU to VAL. The Hemoglobin S gene is then resulted from this and is a rece...
Sickle cell anemia is a hereditary disorder that mostly affects people of African ancestry, but also occurs in other ethnic groups, including people who are of Mediterranean and Middle Eastern descent. More than 70,000 Americans have sickle cell anemia. And about 2 million Americans - and one in 12 African Americans - have sickle cell trait (this means they carry one gene for the disease, but do not have the disease itself).
Sickle cell anemia is an inherited disease of red blood cells. Normally red blood cells contain a protein called hemoglobin A, which carries oxygen to all the organs in the body. With sickle cell anemia, however, the body makes a different kind of protein, called hemoglobin S.
Sickle cell anemia is a genetic disorder. For a person to have sickle cell anemia they must have received two copies of the gene, one from each parent. A person with the sickle cell trait only received one sickle cell gene and a normal hemoglobin gene. People who have the sickle cell trait do not develop sickle cell anemia however they can pass the deformed gene on to their children. People with just the trait do not face the complications caused by the disorder.
Sickle Cell Anemia is an inherited blood disorder that is extremely challenging to live with. Its symptoms are many, with the most prominent being severe pain that can become unbearable to the point where hospitalization is required. Because sickle cell is a genetic disorder, a person is born with it and it is usually permanent. Unfortunately, there are risks and complications associated with this disease. However there are various treatment options for a patient with sickle cell and also support to help people understand and cope with this challenging disease.
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
LAB REPORT 1st Experiment done in class Introduction: Agarose gel electrophoresis separates molecules by their size, shape, and charge. Biomolecules such as DNA, RNA and proteins, are some examples. Buffered samples such as glycerol and glucose are loaded into a gel. An electrical current is placed across the gel.
As seen on many crime shows and at real-life crime scenes, it is necessary to be able to identify DNA. Most of the time, this is done using a technique known as gel electrophoresis. Gel electrophoresis is a method used to separate the macromolecules that make up nucleic acids, such as DNA and RNA, along with proteins. Gel electrophoresis is significant because it has given scientists insight on what cells cause certain diseases and has led to advancements in DNA and fingerprint identification. My experiment will use gel electrophoresis to compare samples of natural and synthetic food dyes. The background for this experiment broaches the following subjects: inventors, real-world uses, necessary components, separation, and information on food dyes.
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...