BME100 f2016:Group11 W1030AM L4

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Contents

Group 11

Andrea Grio
Andrea Grio
Saul Vidrios
Saul Vidrios
Sonya Baran
Sonya Baran
Jake Perrine
Jake Perrine
Joshua Hsu
Joshua Hsu

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR Reaction mix (Taq DNA polymerase, MgCl2 and dNTP’s), 8 tubes (50 µL each)
  • DNA/ primer mix, 8 tubes (50 µL each)
  • A strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G 11 + Positive control none
G 11 - Negative control none
G 11 1-1 Patient 1, replicate 1 45622
G 11 1-2 Patient 1, replicate 2 45622
G 11 1-3 Patient 1, replicate 3 45622
G 11 2-1 Patient 2, replicate 1 73067
G 11 2-2 Patient 2, replicate 2 73067
G 11 2-3 Patient 2, replicate 3 73067


DNA Sample Set-up Procedure

1. Extract the DNA sample and place it into PCR tube

2. Add primer 1 and primer 2 to the PCR tube

3. Add nucleotides to the PRC tube

4. Add DNA Polymerase to the PCR tube

5. Place the PCR tube into the thermal cycler

OpenPCR program

Heated Lid: 100°C

Initial Step: 95°C for 2 minutes

Number of Cycles: 25 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

The main function of DNA Polymerase is to replicate DNA nucleotides. However, PCR (Polymerase Chain Reaction), is to amplify the replication by a larger amount for study. DNA is difficult to isolate already and when the goal is to try and isolate a specific part of the DNA sequence the process becomes more trouble than it's worth. PCR is in short the targeted amplification of a specific strand of DNA. The PCR process has two components an initial and a subsequent reaction, both of which require the usage of 'primers.' Primers are artificially created sequences of DNA that specifically match up with the targeted sequence of DNA designated for amplification.

Primers work in pairs by attaching both to the top strand of DNA as well as the bottom strand. These primers mark where DNA polymerase is suppose to begin replicating the DNA--DNA Polymerase can only replicate existing strands of DNA--since these primers are created in the lab.




SNP Information & Primer Design

Background: About the Disease SNP

Single Nucleotide Polymorphism, SNP, is a disease that is genetically carried by a single point mutation. This SNP disease, with the numerical position of 113347751, is found in homo-sapiens and is located in Chromosome #4. Clinical significance of SNP are listed as, "pathogenic allele," which is defined as one of two/more alternative dorms of a gene that arises by a mutation and are found at the same place on a chromosome.


Primer Design and Testing

In doing these tests, we found that the forward and reverse primers were able to correctly link up with a full gene sequence. It is known that the reverse primers properly created a mutated gene because the database relayed information for the correct gene sequence rather than denying the sequence altogether. Based on this information, it can be confirmed that the database used only reflects accurate information.

forward and reverse primer
reverse and forward primer


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