BME100 f2014:Group34 L4

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Contents

OUR TEAM

Ana Aragon
Ana Aragon
Xavier Richmond
Xavier Richmond
Victor Huerta
Victor Huerta
Ferrin Thomas
Ferrin Thomas
Morgan Seburn
Morgan Seburn
Teja Vemulapalli
Teja Vemulapalli

LAB 4 WRITE-UP

Protocol

Goal

Plan a Polymerase Chain Reaction (PCR) protocol.

Lab Scenario

Several patients have submitted DNA to be tested for a disease marker. We received three replicate DNA samples from two patients.


Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50μL each: Mix contains Taq DNA polymerase, MgCl_2, and dNTP's
  • DNA/primer mix, 8 tubes, 50μL each: 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 discarded tips
  • Micropipettor
  • OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G34 + Positive control 88697
G34 - Negative control 48246
G34 1-1 Patient 1, replicate 1 88697
G34 1-2 Patient 1, replicate 2 88697
G34 1-3 Patient 1, replicate 3 88697
G34 2-1 Patient 2, replicate 1 48246
G34 2-2 Patient 2, replicate 2 48246
G34 2-3 Patient 2, replicate 3 48246


DNA Sample Set-up Procedure

  1. Transfer collected DNA into special PCR tubes using the micropippetor
  2. Add Primer 1 to the PCR tube
  3. Add Primer 2 to the PCR tube
  4. Add the 4 nucleotides: A, T, G, C, to the PCR tube
  5. Finally add the DNA Polymerase to the PCR tube
  6. Place the PCR tube in the Thermal Cycler


OpenPCR program

Heated lid: 100°C

Initial step: 95°C for 2 minutes

Number of cycles: 35

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

PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. It is comprised of 4 separate components: Template DNA, Primers, Taq Polymerase, and deoxyribonucleotides. Template DNA is the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other. The primers are short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer. Taq Polymerase is a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. This specific type of DNA polymerase is the most commonly used enzyme as it has two characteristics that make it very suitable for PCR. First, it can generate new strands of DNA using a DNA template and primers. Second, they are very heat-resistant, and can therefore withstand the high heat of the PCR. Deoxyribonucleotides (dNTP's) are the single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.


The initialization step heats the reaction to a temperateure of 94-96°C for 1-9 minutes. At the denaturation step the reaction is heated to 95°C, the DNE double helix molecule separates, creating two single stranded DNA molecules.

In the Annealing step the temperature cools down to 57°C, the molecules attempt to pair up, however, the primers make their way in and attach to the molecules before the molecules can reattach.

In the extension step, the reaction is then heated back up to 72°C, activating the DNA polymerase. Once the polymerase finds a primer, it begins to add complimentary nucleotides onto to the strand, falling off once it reaches the end.

the final elongation step keeps the reaction at a temperature of 72°C to ensure any remaining single-stranded DNA is fully extended.

The final hold cools the reaction to 4°C for the short-term storage of the reaction.

This cycle repeats 30 times, yielding over a billion fragments of the target DNA.



Adenine (A) pairs with Thymine (T)

Guanine (G) pairs with Cytosine (C)


DNA base pairing occurs in the extension step and the final elongation step as the temperature is held at 72°C, enough to keep the DNA polymerase activated.


Description of image

"Polymerase chain reaction" by Enzoklop - Own work. Licensed under Creative Commons Attribution-Share Alike 3.0 via Wikimedia Commons - http://commons.wikimedia.org/wiki/File:Polymerase_chain_reaction.svg#mediaviewer/File:Polymerase_chain_reaction.svg





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