BME100 f2015:Group17 8amL4

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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: Sam Stone
Name: Jadyn Ziegler
Name: Mahoro Uwiringiyimana
Name: Jake Feltz
Name: Zachary Ramsey
Name: Nathan Natividad

LAB 4 WRITE-UP

Protocol

Materials

PCR Reaction Sample List

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2

and 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: only use each only once. Never re-use disposable pipette tips or

samples will be cross-contaminated

  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 97780
G# 1-2 Patient 1, replicate 2 97780
G# 1-3 Patient 1, replicate 3 97780
G# 2-1 Patient 2, replicate 1 21836
G# 2-2 Patient 2, replicate 2 21836
G# 2-3 Patient 2, replicate 3 21836


DNA Sample Set-up Procedure

  1. Harvest DNA from host source (only a small amount is required)
  2. Move the extracted DNA into a special PCR tube
  3. Add Primer 1 to the PCR tube
  4. Add Primer 2 to the PCR tube
  5. Add Nucleotides to the PCR tube
  6. Add DNA Polmers to the PCR tube
  7. Place PCR tube into thermocycler and begin reaction

OpenPCR program

  • Heat the lid to 100°C
  • Then lower to 95°C for 2 minutes
  • Let it complete 25 cycles.
  • Denature at 95°C for 30 seconds, anneal at 57°C for 30 seconds, and

extend at 72°C for 30 seconds

  • Finally, lower to 72°C. This will last for 2 minutes
  • The final hold will be 4°C.






Research and Development

PCR - The Underlying Technology
Template DNA: A DNA strand that acts as a template for replicating the complimentary strand. It is used in a PCR reaction in a way so that DNA polymerases can copy the strand whole strand of DNA. This is important because it allows for Primers to copy a certain segment of the DNA. Primers: Short pieces of DNA that are made in a laboratory and designed to match the segment of DNA you want to copy. Its function in a PCR reaction is to heavily focus in synthesizing the requested DNA segment. Taq Polymerase: Used to synthesize a new DNA strand that is made from a template. Its role in a PCR reaction is to copy the same segment of DNA over and over again to amplify the segment. Deoxyribonucleotides: A single unit of DNA that is composed of one nitrogen base, a deoxyribose sugar, and a phosphate group. Its function in a PCR reaction is to provide the building blocks for synthesizing a DNA strand

The Process of Thermal Cycling: First, the DNA is placed in a thermocycler. The thermocycler raises the temperature of the DNA to 95 degrees Celsius for three minutes. After the DNA double helix has been kept at this temperature for this time, a thirty second period then begins in which two single-stranded DNA molecules are created. These two single-strands are a result from the separation of the original double helix. After this process, the DNA is brought down to a temperature of 57 degrees Celsius for thirty seconds. During this time period, primers attach to DNA sites so the two single strands do not bond together again. Once the primers are attached, the thermocycler changes temperature to 72 degrees Celsius for 30 seconds. This temperature change activates DNA polymerase. Finally, after three more minutes of sitting in 72 degrees Celsius, nucleotides are added to the single-stranded DNA by the primers. The primer follows the entire strand to add nucleotides. The temperature then changes to four degrees Celsius to halt the process.

Adenine (A) pairs with Thymine (T). Cytosine (C) pairs with Guanine (G). Guanine (G) pairs with Cytosine (C). Thymine (T) pairs with Adenine (A).


PCR PIC.jpg





SNP Information & Primer Design

Background: About the Disease SNP The two steps of thermal cycling during which base pairing occurs are “Annealing” and the “Extension” stages. During the Annealing stage the temperature is lowered to 57 degrees celsius. At this temperature the DNA strands are looking to recombine however the massive amount of primer 1 (a specific sections of the DNA) present causes the DNA strands to bind to the primers at the top strand at one end of the segment of interest. At the Extension stage, the second primer also binds to the bottom strand at the other end of the DNA strand.

This variation is found in Homo sapiens. The variation is located on the chromosome, 16:89919736. The Clinical significance of this SNP is pathogenic. SNP is associated with the gene, MC1R. MC1R​ stands for Melanocortin 1 receptor. The MC1R gene provides instructions for making a protein. An allele is an alternate form of a gene caused by a mutation. They are found at the same place on a chromosome.

Primer Design and Testing

The objective of this process was to understand how base pairs are altered within the human genome. This includes mutations from unwanted base pairs. The process illustrated what steps are taken when base pairs are changed. This included the change from CGC to CGG.


PCR.JPG