BME100 f2017:Group8 W1030 L4
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LAB 4 WRITE-UP
have the same forward primer and reverse primer
the samples will become cross-contaminated
PCR Reaction Sample List
HEATED LID: 100°C: Hydrogen bonds which hold the DNA strand together are broken to initiate denaturing.
FINAL STEP: 72°C for 2 minutes: DNA polymerase continues to add DNA nucleotides until it reaches the end of the strand. Once the new double-strand is formed, the DNA polymerase falls off.
Research and Development
PCR - The Underlying Technology
When using the PCR method to copy DNA molecules, first, a template DNA molecule needs to be obtained. A specific strand DNA molecule is extracted from a subject, and is used as a template for the PCR process. Next, the primers, which are a small section of DNA nucleotides made in a laboratory, bind to sites on the DNA strands on either end of the segment that needs to be copied. After the primers bind to the DNA, a taq polymerase enzyme assembles the nucleotides into strands of DNA. Finally, the dNTP’s supplies the bases ( dATP, dTTP, dCTP and dGTP) to the polymerase enzyme finalizing the process making a complete strand of DNA.
During the first two minutes, the the DNA is put in 95°C the double helix DNA strand separates into two single-stranded DNA molecules. Then, for 30 seconds at 95°C during DNA denaturation, the double-stranded DNA will separate into two single-strands. Next, for 30 seconds at 57°C the annealing process begins where single-stranded DNA molecules attempt to pair up, but the primers crowd in the tube and lock onto the strand before a double strand can form. During the next process, the DNA extends at 72°C for 30 seconds in which the DNA polymerase is activated, which locates a primer attached to a single-strand of DNA. The DNA polymerase adds complementary nucleotides to the DNA strand until it reaches the end of the strand and falls off. Finally, for two minutes at 72°C the DNA polymerase is stimulated to add complementary nucleotides to the single-strand of DNA by sliding along the single DNA strand. Once completed, the DNA polymerase falls off the newly created double-strand. For later use, the DNA can be stored at 4°C to prevent the it from denaturing.
SNP Information & Primer Design
Background: About the Disease SNP
A single nucleotide polymorphism(SNP) is a variation in the genetic code where a single nucleotide on the same area of chromosome differentiates members of the same species. These mutations have a wide effect on quality of life that spans from life-threatening diseases like sickle-cell anemia to non-threatening disorders like a change in hair color. One specific SNP, found on the Apolipoprotein E (APOE) gene on chromosome 19:44907853, interchanges Cytosine with Thymine and results in Alzheimer's disease. The point mutation that results in this SNP codes for a different codon that results in the creation of a different codon. Because SNPs affect the genetic code of an organism, if the organism matures to reproduction age, these mutations can be passed down and become heritable and specific to a population.
Primer Design and Testing
Because SNPs are a point-mutation in the genetic code, in order to design a primer for the disease, one needs an already sequenced genome of a normal person. To create the forward primer, first one must find the position of the nucleotide that needs to change, then transcribe the preceding nineteen nucleotides to create a twenty nucleotide long strand with the changed-nucleotide at the end. To create the reverse primer, add two hundred to the position of the original code in order to reach the reverse primer, than write the bases from left to right of the bottom strand. To test the primer, one can copy both reverse and forward primer into a non-disease human genome sequence and check to make sure that there are no results. There must be no results since the program tests to check for non-disease where the SNP that was created is for a disease.