BME100 f2015:Group11 8amL4

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Owwnotebook icon.png BME 100 Fall 2015 Home
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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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OUR TEAM

Name: Dean Spyres
PCR Part B
Name: Kyra Temple
Role(s)
Name: Kyle GreenzweigRole(s)
Name: Theodore Valenzuela
Role(s)
Name: Maria ROman
Role(s)
Name: student
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, dNTP's
  • DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discard tips
  • Micropipettor
  • Open PCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G11 + Positive control None
G11 - Negative control None
G11 1-1 Patient 1, replicate 1 24467
G11 1-2 Patient 1, replicate 2 24467
G11 1-3 Patient 1, replicate 3 24467
G11 2-1 Patient 2, replicate 1 87309
G11 2-2 Patient 2, replicate 2 87309
G11 2-3 Patient 2, replicate 3 87309


DNA Sample Set-up Procedure

  1. Pipette the patients DNA into the PCR tube
  2. Add Primer mix to the PCR tube
  3. Add dNTP's to the PCR tube
  4. Add DNA polymerase (Taq DNA)
  5. Now place the PCR tube in to the thermal cycler


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






Research and Development

PCR - The Underlying Technology

Q1. What is the function of each component of a PCR reaction?

Template DNA serves as the template for replication throughout the entire PC reaction. Primers are required to begin the process of DNA replication and are required for selective and repetitive replication. Taq polymerase serves as a copying enzyme that attaches to the template DNA and copies the strand that the primers bond to. Finally, the nucleotides are used to build the copy of the selected template DNA.

Q2. What happens to the components (listed above) during each step of thermal cycling?

AT 95°C the DNA double helix breaks apart into two separate strands. As the PCR tube begins to cool down the DNA strands attempt to rejoin, however, the primers stop this by bonding to their target area on each strand of DNA. Then the temperature is brought up to 72°C. When this happens the DNA polymerase becomes active and looks for primers attached to single DNA strands. The polymerase then uses the dNTP's to add complimentary nucleotides to the single DNA strand and then falls off when it reaches the end of the strand. The DNA is then held at 4°C in order to limit the activity of the Taq polymerase.

Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.

The first step that base pairing occurs at is when the PCR tube begins to cool down from 95°C. At this step the primers bond onto the single strands of DNA. The second step that base pairing occurs at is when the PCR tube is heated back up to 72°C. During this step the polymerase enzyme builds replica DNA with the dNTP's.



Q3. Base Pairing
DNA is composed of the four nucleotides, Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). The DNA is made up of two nucleotide sequences that bind together through hydrogen bonding. The bases have specific bonding tendencies, characterized by Adenine always bonding to Thymine and Cytosine always bonding to Guanine.

SNP Information & Primer Design

Background: About the Disease SNP

Single Nucleotide Polymorphisms is a genetic variation in a single DNA nucleotide. The rs1805008 is most commonly found in humans, and it is located in chromosome number 16. The MC1R gene, also known as melanocortin 1 receptor, determines pigmentation in skin, hair, and eye color. This gene produces two types of melanin: eumelanin and pheomelanin. Individuals that possess eumelanin have darker hair and skin, while individuals with pheomelanin tend to have lighter freckled skin. A variation in this MC1R gene increases the risk of skin cancer such as melanoma. An individual with the variation in the MC1R gene is prone to develop diseases like skin cancer, because MC1R interferes with the production of eumelanin, leading to a lack of skin pigmentation and more exposure to UV rays.


Primer Design and Testing

When we checked the non-disease primer in the database, and the non-disease strand came up with 220bp. The diseased primer did not find any matches. This result was to be expected because the diseased primer had a mutation in the gene; therefore, the codon would not match the non-disease (normal) sequence.To change the non-diseased primer to a diseased primer we only changed one nucleotide base from a C to a T which was on the numerical position 89919736 on the 16th chromosome, this affected the subsequent primers that would all have a changed base pair, leading to a different codon.

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