BME100 f2017:Group13 W1030 L4
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The Nightmare Before BMELAB 4 WRITE-UPProtocolMaterials
Research and DevelopmentPCR - The Underlying Technology PCR Reaction Components and Their Functions One main component of a PCR reaction is the template DNA. The template DNA is the sample of DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other. Primers, another important component in a PCR reaction, are short pieces of DNA made in a laboratory. In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy. Primers are necessary because DNA polymerase can only attach onto an existing piece of DNA. Taq polymerase, an enzyme that is used to bind to the primer sequences, is used to extend the second strand by adding nucleotides. Nucleotides are composed of deoxyribonucleotides, which are the single base units (A, T, G, and C) that act as building blocks for new DNA strands. http://learn.genetics.utah.edu/content/labs/pcr/ Thermal Cycling Procedure The initial step of thermal cycling is to heat up the DNA to its boiling point of 95 degrees Celsius for two minutes so that the DNA unfolds. After denaturing the DNA for 30 seconds, the primers either bind or anneal to complementary matches on the target DNA sequence. The double helix will then separate, creating two single-stranded DNA molecules. Once the single-stranded DNA molecules are created, anneal the DNA at 57 degrees Celsius for 30 seconds. The single-stranded DNA molecules will naturally attempt to pair up, however, there are more primer sequences than DNA strands in the tube. Primers will crowd their way in and lock onto target before strands can rejoin. To activate the DNA polymerase, extend the DNA at 72 degrees Celsius for 30 seconds. Finally, the DNA will locate the primer attached to the single DNA strand and begin to add the complementary nucleotides. This process continues until the polymerase reaches the end of the strand. End the procedure by holding the temperature at 4 degrees Celsius. http://learn.genetics.utah.edu/content/labs/pcr/ Base-Pairing It is during the annealing and extending part of thermal cycling that the base-pairing takes place. Adenine pairs with Thymine (and vice-versa), while Guanine pairs with Cytosine (and vice-versa.) Annealing is when the system is cooled and the target DNA is bound with the short DNA primers. During extension, the Taq polymerase binds to each PCR primer and begins adding in the corresponding nucleotides. https://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/basepair.html SNP Information & Primer DesignBackground: About the Disease SNP SNP is defined as a single nucleotide polymorphism, meaning one of the nucleotide bases (A, T, G, or C) has a common variation among individuals who share the same structure of DNA. Nucleotides are compounds linked to phosphate groups and form the basic structural unit of nucleic acids. SNPs occur in the human genome. The variation, rs769452, is found in Homo sapiens, more specifically, in the chromosome 19:44907853. The clinical significance of this SNP variation is that it is pathogenic and is linked to issues such as Alzheimer's, strokes, and subarachnoid hemorrhage. APOE is defined as Apolipoprotein E. and it is the protein that "binds to a specific liver and peripheral cell receptor and is essential for the normal catabolism of triglyceride-rich lipoprotein constituents." It is an integral part of antioxidant activity, cholesterol, protein binding, and other important functions. When a gene has one of two or more alternative forms, derived from mutations, found in the same place on a chromosome, it is considered an allele. In case of the given SNP, the disease-associated allele contains the codon CTG which is then changed into CCG. Its numerical position is 44907853 (base pairs). https://www.ncbi.nlm.nih.gov/ https://www.thoughtco.com/allele-a-genetics-definition-373460 Primer Design and Testing The non-disease forward primer would be 5'-AGCGGCCAGCGCTGGGAACT-3’. Since every PCR reaction needs two primers, the non-disease reverse primer would be 5’-CAGGCCCCCCAAGACTTAGC-3’, located in the numerical position 44908053. The disease forward and reverse primers would be 5’-AGCGGCCAGCGCTGGGAACC-3’, and 5’-CAGGCCCCCCAAGACTTAGC-3’, respectively.
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