BME100 s2016:Group1 W1030AM L4

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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

Eman Alawi A Abu AlrahiRole(s)
Eman Alawi A Abu Alrahi
Role(s)
Kyler BlairRole(s)
Kyler Blair
Role(s)
Carson KennedyRole(s)
Carson Kennedy
Role(s)
Richard LiRole(s)
Richard Li
Role(s)
Nicole PenroseRole(s)
Nicole Penrose
Role(s)
Cami RowanRole(s)
Cami Rowan
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, 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 tube
  • 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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 46191
G# 1-2 Patient 1, replicate 2 46191
G# 1-3 Patient 1, replicate 3 46191
G# 2-1 Patient 2, replicate 1 81527
G# 2-2 Patient 2, replicate 2 81527
G# 2-3 Patient 2, replicate 3 81527


DNA Sample Set-up Procedure

  1. Lid heated to 100 degrees Celsius
  2. Initial: Thermal cycler is set to 95 degrees Celsius for 2 minutes
  3. Run for 25 cycles
  • Denature for 30 seconds at 95 degrees Celsius
  • Anneal for 30 seconds at 57 degrees Celsius
  • Extend for 30 seconds at 72 degrees Celsius
  1. Final: Thermal cycler is set to 72 degrees Celsius for 2 minutes
  2. Final Hold: Thermal cycler is at 4 degrees Celsius

OpenPCR program






Research and Development

PCR - The Underlying Technology

Research and development - the underlying technology PCR works by heating and cooling DNA. Initial DNA is placed into a container where primers are added. Primers bind to specific segments of interest once the DNA is separated in heating. When DNA is heated to the specific heat of 95 degrees celsius, DNA begins to unwind and separate. When the DNA strands become single stranded, the solution is cooled to a temperature of 50 degrees celsius.

As the temperature cools, DNA will naturally want to rejoin due to the electrical charges on both strands but before this happens primers interrupt this process through a manner similar to competitive inhibition and manage to bind onto the strands of DNA before they can reform. With primers bound onto the DNA strand the process of DNA replication can begin. The solution is then heated to 72 degrees celsius in order for DNA polymerase to activate. DNA polymerase is an enzyme that binds to specific primer regions and begins adding nucleotides to the strand until it reaches the end.

This process is repeated several times until around cycle three when the desired targeted DNA segments are produced. This is a result of DNA strands with both primer one at one end and primer two at one end. This is a result of successive replication of DNA at specific primer points through several cycles. As this process continues with these desired targeted DNA segments also multiplying the results quickly become pronounced and in abundance. One of the most interesting aspects about PCR found in research was the specificity of the DNA polymerase. DNA polymerase is destroyed at high temperatures except for a specific type found in a strain of bacteria called Thermus Aquaticus. This polymerase is referred to as Taq. A side note, Taq is a powerful example of the implications of patenting a DNA strand as its manufacture and ownership has been contested several times in court. Because Taq does not degrade at high temperatures, its usage is incredibly important in PCR as it can survive the changing heat (which has powerful implications on its value).




SNP Information & Primer Design

Background: About the Disease SNP SNP is a short genetic variation which a specific sequence of DNA differs by a single base pair, from another similar sequence. SNPs are found in all life forms with DNA. This can affect physical appearance, susceptibility to disease, and response to medication of the life form.

Once a region of DNA has been selected, a primer can be designed to amplify the DNA. Heat is used to separate the DNA strands. Primer 1 and primer 2 attach to the free stands and are built upon with nucleotides, and the cycle repeats until there are billions of copies of the DNA sequence.

To design a primer it must be able to bind with the target DNA. The primer consists of 18-22 base pairs, primer 1, and a reverse sequence called primer 2. The SNP should be located on the end of the primer so that it becomes amplified.


Primer Design and Testing




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