BME100 s2015:Group8 9amL4

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Contents

OUR TEAM

Name: An TranRole(s)
Name: An Tran
Role(s)
Name: Molly GolekRole(s)
Name: Molly Golek
Role(s)
Name: Anthony GambirazioRole(s)
Name: Anthony Gambirazio
Role(s)
Name: Austin LehewRole(s)
Name: Austin Lehew
Role(s)
Name: Robert CulibrkRole(s)
Name: Robert Culibrk
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat
  • Disposable Gloves
  • PCR Reaction Mix, 8 tubes, 50 uL each: Mix contatins Taq DNA polymerase, MgCL2, and dNTP's
  • DNA/primer mix, 8 tubes, 50 uL 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 machines: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G8 P Positive control none
G8 N Negative control none
G8 1-1 Patient 1, replicate 1 74239
G8 1-2 Patient 1, replicate 2 74239
G8 1-3 Patient 1, replicate 3 74239
G8 2-1 Patient 2, replicate 1 82959
G8 2-2 Patient 2, replicate 2 82959
G8 2-3 Patient 2, replicate 3 82959


DNA Sample Set-up Procedure

  1. Acquire the DNA samples from the patients as well as PCR reaction mix
  2. Label all of the PCR tubes for all of the samples that will be tested
  3. Place a new pipette tip on the micropipettor
  4. Take 50 microliters of the PCR reaction mix
  5. Transfer PCR reaction mix to the corresponding labeled empty PCR tube
  6. Discard the used pipette tip
  7. Put a new pipette tip on the micropipettor
  8. Take 50 micro liters of patient DNA sample/primer corresponding to the label on the tube
  9. Transfer the patient DNA into the tube containing the PCR reaction mix
  10. Repeat steps 3 through 9 for all samples. Be sure to place the DNA in the correct labeled tubes
  11. Place the tubes 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
  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
Often, a phenotype such as a deformity or mutation is a result of a specific section of DNA. Unfortunately, there are many technological and practical limitations to analyzing the entire sequence of a DNA strand in search for the specific section that generates the phenotype. Primarily, obtaining the amount of DNA necessary to confirm the presence of a specific sequence is difficult, simply because a lot of DNA is required in that case. PCR, or polymerase chain reaction, eradicates this problem by allowing for a cheap and effective way to replicate or “amplify” a specific sequence of DNA: if the specific section is present in the given DNA sequence, the section will continue replicating until there are roughly over a billion copies, undoubtedly making it much easier to detect its presence.

The following are the functions of assorted portions of a PCR reaction: the template DNA is the original DNA in which a specific section or sequence is targeted. The primers are essentially the beginning and end nucleotides of the targeted sequence. The Taq Polymerase attaches to a single strand of DNA, reads each nucleotide, and then attaches the appropriate base pairs. The deoxyribonucleotides are the base pairs that Taq polymerase attaches to the newly created single strand of DNA in order to complete the strand.

The purpose of heating the DNA to 95 degrees Celsius for three minutes is to ensure that the DNA is unwinding from its usual double-helix shape and is ready for the denaturing step of PCR. Next, when the temperature is maintained at 95 degrees Celsius for an additional thirty seconds, the denaturing process actually occurs: the two strands of DNA completely unwind and split. During the subsequent step, the “annealing”, where the temperature is decreased to 57 degrees Celsius for thirty seconds, the two strands naturally have a proclivity for rejoining. The “Extend” step allows for the Taq polymerase to take action, joining the appropriate base pairs and amplifying a specific sequence with the assistance of primers. In the FINAL STEP, the isolated segments of DNA are allowed to solidify, and return to their natural state (double-helix formation). Lastly, the FINAL HOLD portion allows for the sections of DNA to be cooled adequately for storage.

The base pairing is as follows: Adenosine binds to Thymine, while Cytosine binds to Guanine.

The process of base pairing begins in the “annealing” step. The temperature at this step allows for base pairs to begin to come together naturally. Base pairs, which exist in a lock-and-key format, ensure that only the appropriate base pairs bind to the isolated strand. The temperature of the “extend” step, 72 degrees Celsius, is when the Taq polymerase is binding base pairs at its fastest rate.

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