BME100 f2015:Group4 1030amL4

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Contents

OUR TEAM

Name: Gabrielle Mills
Name: Gabrielle Mills
Name: Christa Deckman
Name: Christa Deckman
Name: Neil Rastogi
Name: Neil Rastogi
Name: Ngan Nguyen
Name: Ngan Nguyen
Name: Evan Higgs
Name: Evan Higgs
Name: Dylan Bunch
Name: Dylan Bunch

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Gloves (disposable)
  • PCR reaction mix (contains Taq DNA polymerase, magnesium (II) chloride, and dNTP); 8 tubes; 50 microliters each

(http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)

  • DNA primer mix (each has a different template DNA, but the same forward and reverse primers); 8 tubes; 50 microliters each
  • Strip of empty PCR tubes
  • Pipette tips (disposable)
  • Cup in which to put discarded tips
  • Micropipette
  • OpenPCR machine (for every two groups)

(https://myasucourses.asu.edu/bbcswebdav/pid-12212866-dt-content-rid-62446597_1/courses/2015Fall-T-BME100-81997-81998/BME100LabWorkbookDNALabsFa2015.pdf)

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G4 P Positive control none
G4 N Negative control none
G4 1-1 Patient 1, replicate 1 12188
G4 1-2 Patient 1, replicate 2 12188
G4 1-3 Patient 1, replicate 3 12188
G4 2-1 Patient 2, replicate 1 54737
G4 2-2 Patient 2, replicate 2 54737
G4 2-3 Patient 2, replicate 3 54737

(https://myasucourses.asu.edu/bbcswebdav/pid-12212866-dt-content-rid-62446597_1/courses/2015Fall-T-BME100-81997-81998/BME100LabWorkbookDNALabsFa2015.pdf)

DNA Sample Set-up Procedure

  1. Go to your T.A. and get the patient's IDs. Write those ID’s down on separate piece of paper as well as in your table.
  2. Create labels for your tubes:
    1. G4 1-1
    2. G4 1-2
    3. G4 1-3
    4. G4 2-1
    5. G4 2-2
    6. G4 2-3.
  3. Move extracted DNA from patient who test positive and the patient who tested negative for SNP and place the DNA samples into the respective PCR tubes.
  4. Add Primer 1 to all Patient 1 and Patient 2 labeled PCR Tubes
  5. Add Primer 2 to all Patient 1 and Patient 2 labeled PCR Tubes
  6. Add Nucleotides(A’s, C’s, G’s, T’s) to all Patient 1 and Patient 2 labeled PCR Tubes
  7. Add DNA Polymerase to all Patient 1 and Patient 2 labeled PCR Tubes:
  8. Now that all the PCR tubes are filled up, place them into the DNA thermal cycler, close the lid, then press start.


OpenPCR program

  • Heated Lid: 100 degree Celsius
  • Initial Step: 95 degree Celsius for two minutes
  • Number of Cycles: 25--Denature at 95 degree Celsius for thirty seconds--Anneal at 57 degree Celsius for thirty seconds--Extend at 72 degree Celsius for thirty seconds
  • Final Step: 72 degree Celsius for two minutes
  • Final Hold: 4 degree Celsius

(https://myasucourses.asu.edu/bbcswebdav/pid-12212866-dt-content-rid-62446597_1/courses/2015Fall-T-BME100-81997-81998/BME100LabWorkbookDNALabsFa2015.pdf)

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http://learn.genetics.utah.edu/content/labs/pcr/



Research and Development

PCR - The Underlying Technology

The Definitions and Functions of the Parts of PCR Reactions

PCR provides a useful tool for replicating DNA in a timely and efficient fashion. In this PCR reaction, the template DNA is copied into the new DNA. The primers added to the PCR reaction attach to the end of the DNA segment of interest using base-pairing of DNA; the Taq Polymerase then attaches to the primers at the end of the segments. This Taq Polymerase makes a copy of the DNA segment by assembling nucleotides. Finally, the dNTP's (or deoxyribonucleotides) serve as the foundation of DNA segments, built into certain sequences of the four types of nucleotides: Thymine (T), Adenine (A), Cytosine (C), and Guanine (G). This whole process builds new copies of sequences of DNA. (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)

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What Occurs During Thermal Cycling

During the initial step of PCR, the thermal cycler is heated to 95 degree Celsius for three minutes. Inside the tube, the enzymes added to the solution (Taq Polymerase and primers) are turned on. This step heats the DNA for the next step of denaturization, causing them to start unwinding. In the next step, denaturing occurs when the thermal cycler heats to 95 degree Celsius for thirty seconds. During this short period of time, the double strands of DNA are separated into two strands since the heat causes the hydrogen bonds to break. The third step, annealing, occurs at 57 degree Celsius for thirty seconds; during this period, the introduced primers bind to the strands of DNA before the single strands can recombine at this lower temperature. Two primers attach to the end of each strand, one at the beginning and the other at the end. In the fourth step, extension, the thermal cycler is heated to 72 degree Celsius for thirty seconds. In this step, the Taq Polymerase attaches to the primer and uses base pairing of nucleotides to create a copy of the DNA segment until this enzyme reaches the end of the segment. During the final step of the reaction at 72 degree Celsius for three minutes, the reaction guarantees that every piece of DNA was extended in the previous step. Lastly, the final hold at four degree Celsius stops the PCR reaction by deactivating the Taq Polymerase through lowering the temperature. (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)

How the Bases Anneal

The base Adenine (A) bonds to Thymine (T). Thymine (T) attaches to the base Adenine (A). Cytosine (C) binds to Guanine (G). The base Guanine (G) attaches to Cytosine (C). (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)

The Steps of Thermal Cycling in which Base-Pairing Occurs

In thermal cycling of PCR reactions, base-pairing occurs during the steps of annealation and extension. During the step of annealation, the temperature in the thermal cycler drops, prompting the primer to bind to DNA segment of interest. Next, the thermal cycler raises the temperature, causing the Taq Polymerase to attach to the primer. It then uses base pairing on the DNA segment to create a copy of the original DNA. (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)




SNP Information & Primer Design

Background: About the Disease SNP

This specific disease SNP “TGG” affects the production levels of melatonin. A nucleotide substitution from CGG to TGG, causes for the gene MC1R to amplify its function, which is to encode the receptor proteins for melanocyte hormones. These receptor proteins help facilitate the production of melatonin. Due to this amplification, melatonin levels (pheomelanin) increase which affect the phenotype of individuals with this disease-related SNP causing for lighter hair and/or skin color. This affected gene is an essential determinant in human pigmentation.


Primer Design and Testing


The results of the primer test demonstrated that a change in a single nucleotide resulted in no matches. Having no matches means that the specific portion of the DNA that we are attempting to replicate can be replicated. That it has no matches also establishes that no other extraneous portions of the DNA are replicated. This means that it establishes a uniqueness between the primer and the DNA strand. This is positive because it will only replicate the targeted SNP rather than extraneous DNA. This specialization of the primer is used to exponentially replicate the SNP using PCR.

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