BME100 f2015:Group13 1030amL4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Drew Worman
Name: Drew Worman
Name: Bramuel Simiyu
Name: Bramuel Simiyu
Name: Tanner Ivey
Name: Tanner Ivey
Name: Jonah Brosemann
Name: Jonah Brosemann
Name: Kaylee Antill
Name: Kaylee Antill
Name: Christopher Chen
Name: Christopher Chen

LAB 4 WRITE-UP

Protocol

Materials

Lab coat
Disposable gloves
8 50 μL tubes of Taq DNA polymerase
8 50 μL tubes of Magnesium chloride
8 50 μL tubes of deoxynucleotide triphosphate
8 50 μL tubes of
8 50 μL tubes of DNA/ Primer mix
Strip of empty PCR tubes
Disposable pipette tips
Cup for discarding tips
Micropipettor
OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G13 + Positive control none
G13 - Negative control none
G13 1-1 Patient 1, replicate 1 21939
G13 1-2 Patient 1, replicate 2 21939
G13 1-3 Patient 1, replicate 3 21939
G13 2-1 Patient 2, replicate 1 73606
G13 2-2 Patient 2, replicate 2 73606
G13 2-3 Patient 2, replicate 3 73606


DNA Sample Set-up Procedure

  1. Place a sample of patient DNA into a PCR tube (carefully label the tube to avoid mixing up samples).
  2. Add the first primer to the PCR tube.
  3. Add the second primer to the PCR tube.
  4. Add the deoxyribonucleotides to the PCR tube.
  5. Add the taq DNA polymerase to the PCR tube.
  6. Put the PCR tube into the thermal cycler (repeat process for each additional DNA sample).

OpenPCR program

  • HEATED LID: 100°C
  • INITIAL STEP: 95°C for 2 minutes
  • NUMBER OF CYCLES: 25
    • Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and
    • Extend at 72°C for 30 seconds
  • FINAL STEP: 72°C for 2 minutes
  • FINAL HOLD: 4°C

Images from Polymerase Chain Reaction (PCR) video <https://www.youtube.com/watch?v=2KoLnIwoZKU>





Research and Development

PCR - The Underlying Technology

Question 1:What is the function of each component of a PCR reaction? The PCR reaction can be broken up into four main components each with a specific and crucial function. The four components are: Template DNA, Primers, Taq Polymerase, and Deoxyrbonucleotides (dNTP's). The function of template DNA is a mechanical guide used to find desired patterns in DNA replication. The function of the primers is to attach to the split DNA and are the starting points for the copying sequence. The function of Taq Polymerase is to identify nucleotides and essentially copy and paste them in the corresponding positions the dNTP's are the nucleotides that are paired after the primers have attached and the Taq polymerase identifies their positions.

Question 2:What happens to the components during each step of thermal cycling? The components each have a part to play throughout the thermal cycling process. In the first step of the cycle referred to as the initial step, all of the components are heated to 95 degrees celsius and during this period of three minutes the DNA begins to unwind so that the two strands do not overlap. The second step is referred to as denaturing and it is when the DNA splits in half. For this to happen successfully the test tube must remain at 95 degrees celsius for an additional thirty seconds. The third step is annealing which is when the primers connect to the split DNA. The DNA attaches to primer because there are many times more primers than in the solution, this helps make sure that the DNA does not reconnect. The third step is called extension. This is the step in which the Taq Polymerase attach to the primers. The final step of the cycle is the replication of the corresponding nucleotides on the template DNA.This cycle is repeated at least three times to produce the desired segment. Once all of this is done the solution is then chilled to 4 degrees celsius to stop the cycle.

Question 3:DNA NucleotidesThe Adenine nucleotide(A) anneals to Thymine nucleotide(T) and Thymine anneals to Adenine. Cytosine (C) binds with Guanine (G) and Guanine binds with Cytosine. A-T, T-A, C-G, G-C

Question 4: During which two steps of thermal cycling does base-pairing occur? Base-pairing occurs during the processes of Annealing and Extending. During annealing, the system is cooled to 57 degrees and the target DNA is binded with short DNA primers that become the starting positions for replicating the desired DNA. During Extention the taq polymerase attach to the primers and begin matching the nucleotides in order to replicate the desired target DNA sequences. These two critical parts during thermal cycling contribute to the adding of base-pairs so that the replication of the desired DNA can take place.





SNP Information & Primer Design

Background: About the Disease SNP

The Nucleotide contains four compounds that make up the basic foundation of DNA. Polymorphisman is the occurrence in which DNA is varied. The species this variation is found in is homo sapiens. The chromosome that the variation is located in is 16:89919736. The clinical significance is that the disease is pathogenic. The disease is associated with the MC1R gene. This SNP is linked to Melanoma. MC1R stands for melanocortin 1 receptor. This has several functions. These include hormone, protein, ubiquitin protein ligase binding, and G-protein coupled peptide , melanocortin, and melanocyte-stimulating hormone receptor activity. An allele is an alternative form of a gene. The disease-associated allele contains the sequence TGG.The numerical position of the SNP is 89,919,736.

Primer Design and Testing The non-disease forward primer is 5'- CAGCATCGTGACCCTGCCGC. The numerical position exactly 200 bases to the right of the disease SNP​ is 89,919,936. The non-disease reverse primer 5'-CTTGTGGAGCCGGGCGATGC. The disease forward primer is 5'- CAGCATCGTGACCCTGCCGT. The disease reverse primer is 5'-CTTGTGGAGCCGGGCGATGA.

Inputting the non-disease primers into the USC In-Silico PCR website gave a 220 bp sequence matching that of the chromosome identified earlier. Inputting the diesease primers, however, resulted in no matches. This result is probably due to the fact that disease primers would not be a natural part of a healthy human genome.

Non-disease primers:

Disease primers

Images from UCSC Genome Bioinformatics Site <http://genome.ucsc.edu/cgi-bin/hgPcr?command=start>




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