BME100 f2015:Group2 1030amL4

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Contents

OUR TEAM

Name: Gage SchrantzRole(s)
Name: Gage Schrantz
Role(s)
Name: Amar JoshiRole(s)
Name: Amar Joshi
Role(s)
Name: Anton VoronovRole(s)
Name: Anton Voronov
Role(s)
Name: Cindy CrockettRole(s)
Name: Cindy Crockett
Role(s)
Name: Lydia ChenRole(s)
Name: Lydia Chen
Role(s)
Name: Colleen RiceRole(s)
Name: Colleen Rice
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix
  • 8 tubes, 50 μL each
  • Mixture: contains Taq DNA polymerase, MgCl​, 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 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 machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control none
G2 - Negative control none
G2 1-1 Patient 1, replicate 1 22993
G2 1-2 Patient 1, replicate 2 22993
G2 1-3 Patient 1, replicate 3 22993
G2 2-1 Patient 2, replicate 1 85311
G2 2-2 Patient 2, replicate 2 85311
G2 2-3 Patient 2, replicate 3 85311


DNA Sample Set-up Procedure

  1. Fill bucket with Ice
  2. Place the Strip of empty PCR test tubes on Ice
  3. Place extracted DNA into the PCR tube
  4. Add primer 1 to the PCR test tube
  5. Add primer 2 to the PCR test tube
  6. Place extra nucleotides in the PCR test tube
  7. Place DNA Polymerase in the PCR test tube
  8. Place tubes into the thermal cycler


OpenPCR program

PCR is a method of copying DNA. PCR's, or Polymerase Chain Reaction, first reaction works by initially yielding twice the amount of molecules of one chain of DNA and then during the second reaction the molecule amount in reaction one is doubled again. The goal of the PRC to replicate specific strands of DNA that are of interest, also known as amplification.


The values below will be used to run heating and cooling programs on the thermal cycler

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
  • 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 concept of Polymerase Chain Reaction(PCR)

     PCR is a very accurate, and convenient method of replicating a few strands of DNA on a mass scale. In other words, while only imputing a few DNA strands, using this method, you can end up with thousands of copies of that DNA strand. 

Functions of each component of a PCR reaction

    Template DNA: The template DNA represents the original strand of DNA. The synthesis of copies of the DNA are based off of the template DNA.
    Primer: String of nucleotides that attaches itself to the template strand. Specifically, it attaches to the beginning of the template strand using the mechanism of base pairing. 
    Taq Polymerase​: it's to run the PCR at high temperature (~60°C and above), which facilitates high specificity of the primers and reduces the production of unspecific product.
    Deoxyribonucleotides(dNTP’s): Four important elements, which are Adenine, Thymidine, Cytosine and Guanine, abbreviated A, T, C and G, to consist of the DNA.​

Q2 (Thermal Cycle). What happens to the components (listed above) during each step of thermal cycling?

INITIAL STEP: 95°C for 3 minutes: The test tube is heating up

Denatureat 95°C for 30 seconds: the template DNA double helix separates creating 2 single stranded DNA molecules

Annealat 57°C for 30 seconds: The single stranded DNA molecules attempt to pair up, there are a lot more primers than DNA strands so the primers lock onto the target before the strands can rejoin.

Extendat 72°C for 30 seconds: DNA polymerase is activated and it locates a primer and attaches on to the end of it, it begins to add complementary nucleotides onto the strand. It continues until it gets to the end of the strand then falls off. The previous cycle repeats.

FINAL STEP: 72°C for 3 minutes: it cools down as the cycle keeps on going

FINAL HOLD: 4°C: kept at a stable temperature.


Base Pairing

   When it comes down to DNA base pairing there are a few simple rules when it comes down to the pairing of bases to each other
   Adenine
      -Adenine always pairs with Thymine
   Thymine
      -Thymine always pairs with Adenine
   Cytosine
      -Cytosine always pairs with Guanine
   Guanine
      -Guanine always pairs with Cytosine

The two steps during thermal cycling that allow base pairing to occur:

 Primer annealing, and extension are the two steps when thermal cycling causes base pairing to occur.

During primer annealing, the mixture containing the template DNA and the primers is cooled down. This cooling allows the primers to bind to their complimentary sequence in that is located in the template DNA. During extension, the mixture is heated. Once the mixture reaches the optimal temperature of 72 degrees C, the DNA polymerase is now able to extend the primers and add the nucleotides onto the primers.

References

   http://www.contexo.info/DNA_Basics/Primers.htm
   http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-T/template.html
   http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BasePairing.html
   http://www.uvm.edu/~cgep/Education/PCR.html



SNP Information & Primer Design

Background: About the Disease SNP SNP is defined as a single nucleotide polymorphism. Two important factors are nucleotides and polymorphisms. A nucleotide is one of the structural components of both DNA and RNA and a polymorphism is the most common type of genetic variation among people. SNP is found in homosapiens in the 16:89919736 chromosome. This mutation is pathogenic and results parkinson.



Primer Design and Testing The results of the primer test indicated the non disease forward primer and its reverse. It presented strands of DNA that were muted and what this mutation would cause. This mutation would result in SNP, or as previously stated single nucleotide polymorphism. Ever PRC reactions needs two primers to demonstrate this in the DNA, this position would be known as dbSNP 144.



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