Essentially, this apparatus performs Polymerase Chain Reaction (PCR). PCR is a reaction that is used to copy DNA segments. PCR is used every day to diagnose diseases, identify bacteria and viruses, and match criminals to crime scenes. Using a heater, it splits the DNA into two segments and allows the PCR reaction to take place. The apparatus raises and lowers temperatures over specific time sequences to control the rate of the reaction. Because of this, the apparatus can be set to perform a specific amount of cycles of PCR.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine's screen turned off.
When we unplugged the white wire that connects (part 6) to (part 2), the machine displayed the incorrect temperature.
Test Run
Machine was first tested on 3/19/14. It appeared to be fully functional. The screen is working and it reached cycle 19 of 35 before we shut it off. After watching the temperature readout change on the screen it appeared that the temperature was fluctuating properly and everything seemed to be working.
Protocols
Thermal Cycler Program
DNA Sample Set-up
disease DNA Tube label:DD
ID:51225 Tube label:11
ID:51225 Tube label:12
ID:51225 Tube label:13
non-disease DNA Tube label:NDD
ID:60779 Tube label:21
ID:60779 Tube label:22
ID:60779 Tube label:23
DNA Sample Set-up Procedure
Step 1: Get patient ID's
Step 2: Set up 8 total reactions. Decide how each PCR reaction tube will be labeled and enter this information in the blank spaces above. Each Tube Label should be unique and have no more than 3 characters.
Step 3: Enter the information into lab report.
PCR Reaction Mix
What is in the PCR reaction mix?
- Taq DNA polymerase, MgCl2, and dNTP's
DNA/ primer mix
What is in the DNA/ primer mix?
- Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
Research and Development
PCR - The Underlying Technology
Q1. What is the function of each component of a PCR reaction?
During a PCR reaction the template DNA is the strand of DNA that will be used as a template to transcribe the messenger RNA for protein synthesis. Primers are used to prime the nucleic acid template for the attachment of the polymerase. This is the first step towards duplicating that template. The primer directs the polymerase to move in a 5' to 3' direction (drawn left-to-right) because of the 'direction' of DNA. Taq DNA Polymerase from Thermus aquaticus is a thermostable DNA polymerase that is used for the polymerase chain reaction (PCR) in order to amplify DNA sequences. Nucleotides (dNTPs or deoxynucleotide triphosphates) are single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.
Q2. What happens to the components (listed above) during each step of thermal cycling?
The first step in PCR is to heat the mixture to a high temperature, usually 94 to 95 °C, for about five minutes. The hydrogen bonds that hold together the two strands of a double helix are broken at these temperatures, and the DNA separates into single strands. This process is termed denaturation. In the second step, the PCR mixture is cooled to a lower temperature, typically between about 50 °C and 65 °C. This allows the primers to anneal to their specific complementary sequences in the template DNA. The temperature for this step is chosen carefully to be just low enough to allow the primers to bind, but no cooler. In the third step, the reaction is heated again, usually to about 72 °C, the temperature at which the DNA polymerase is most active. At 72 °C, Taq DNA polymerase adds nucleotides to the 3′ ends of annealed primers at the rate of about two thousand nucleotides per minute. Therefore, to amplify a sequence that is one thousand nucleotides long, the primer extension step must last about thirty seconds at 72 °C. By the end of this step, each template strand has a new complementary strand. This completes the first cycle of the PCR reaction.
Q3.Which base anneals to each base listed below?
Adenine(A): Thymine
Thymine(T): Adenine
Cytosine(C): Guanine
Guanine(G): Cytosine
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)
BME 100 Spring 2014
