BME100 f2015:Group3 1030amL4

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OUR TEAM

Name:
James Wood
Name:
Jennifer Le
Name:
Shannon Grassi
Name:
Noah Pollack
Name:
Lauren Butler
Name:
Carlos Garrido

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix, 8 tubes (50 μL each): Mix contains Taq DNA polymerase, MgCl2, 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
  • Strip of empty PCR tubes
  • Dispoable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G3 1-1 Patient 1, replicate 1 45247
G3 1-2 Patient 1, replicate 2 45247
G3 1-3 Patient 1, replicate 3 45247
G3 2-1 Patient 2, replicate 1 78801
G3 2-2 Patient 2, replicate 2 78801
G3 2-3 Patient 2, replicate 3 78801


DNA Sample Set-up Procedure

  1. Use a micropipette to move the extracted DNA into the PCR tube
  2. Use a micropipette to add Primer 1 to the PCR tube
  3. Use a micropipette to add Primer 2 to the PCR tube
  4. Use a micropipette to add nucleotides to the PCR tube
  5. Use a micropipette to add DNA polymerase to the PCR tube
  6. Place the PCR tube into a DNA thermal cylinder


OpenPCR program


BMETC.PNG





Research and Development

PCR - The Underlying Technology

What is the function of each component of a PCR reaction?
In a PCR experiment, the template DNA serves as a blueprint for the replication of other DNA's. In the PCR process, primers attach themselves to match a desired segment of DNA on the template DNA through complementary base pairing. Primers are shorter pieces of DNA that are custom made in laboratories and thus can have any sequence of nucleotides desired, and there are 2 primers needed to replicate the denatured DNA: one primer to attach to the top DNA strand and a second primer for the bottom strand. After the primers are attached to the desired segment of DNA, the enzyme Taq DNA polymerase attches itself to the other end of the primers; DNA polymerase functions to assemble nucleotides into new strands of DNA from the template DNA. Throughout PCR, Deoxyribonucleotides (dNTP’s)--a single unit of DNA comprised of a nitrogenous base (A,C,G,T), a deoxyribose sugar, and a phosphate group--serves as a coding monomer for DNA​.

What happens to the components (listed above) during each step of thermal cycling?
During the initial step of PCR, the DNA thermal cycler raises its temperature to 95°C for 3 minutes, which is almost boiling. This is done in order to allow enough time to denature, or separate, the template DNA's double helix structure, creating two single-stranded DNA molecules; denaturing DNA at 95°C will last for 30 seconds. After the DNA is denatured, anneal occurs and the temperature is lowered to 57°C for an additional 30 seconds. At this temperature, primers work quickly to bind to the the DNA strand's deoxyribosenucleotides on the nucleotide compenents to prevent the single-stranded DNA molecules from pairing up. Then the temperature is raised up to 72°C for 30 seconds for the Taq DNA polymerase to locate a primer attached to a single DNA strand. In the final step of PCR, the temperature is held at 72°C for 3 minutes for the DNA polymerase to add complementary nucleotides onto the strand; it continues to extend until it reaches the end of the DNA strand and falls off. After the desired amount of cycles are complete, a final hold at 4°C is done in order preserve and store the newly created DNA, but also to prevent the taq polymerase from further replicating anymore nucleotides, thus ending the PCR process.

Which base anneals to each base listed below?

Nucleotide Base Annealing Nucleotide Base
Adenine (A) Thymine (T)
Thymine (A) Adenine (A)
Cytosine (C) Guanine (G)
Guanine (G) Cytosine (C)

As the table above illustrates, T anneals to the base A, A anneals to the base T, G anneals to the base C, and C anneals to the base G.

During which two steps of thermal cycling does base-pairing occur?
A base pair consists of a complementary purine (Adenine, Guanine) and pyrimidine (Thymine, Cytosine) being attached to each other; the pairings are A-T or C-G. Base pairing in thermal cycling of PCR occurs at the anneal, extend, and final step.
The anneal step is when the temperature is lowered to 57°C and the primers attach themselves to the specified nucleotides of the template DNA through base pairing. The extend step in the PCR process follows anneal; it is when the temperature is raised up to 72°C for 30 seconds; this allows for the Taq DNA polymerase to locate the primers attached to the DNA and then attach itself from where the primers left off, which is done through base-pairing. The final step in which the temperature is held at 72°C for 3 minutes is to allow the DNA polyermase to perform its function. The DNA polymerase, during this step, begins to add complementary nucleotides to make a new strand off of the DNA template. The extend process also uses base-pairing in order to create a new single-stranded DNA molecule because the DNA polymerase creates complementary base pairs to the template DNA's base pairs and pairs them.

Illustration of primers and Taq polymerase

explanation image 1

After the desired amount of cycles have been preformed it is common to drop the temperature of the samples back down to the original base temperature of approximately 4˚C to prevent potential non-specific binding by limiting the activity of Taq polymerase.

Citation for reference of images listed above:



SNP Information & Primer Design

Background: About the Disease SNP SNP stands for Single Nucleotide Polymorphism. It occurs within a species or paired chromosome and causes a difference in one single nucleotide, Ex: CCTA to CGTA. Basically, they show the genetic variation between individuals. Within these variations determine how humans (or other species) interact with certain pathogens, chemicals, or drugs. SNPs are essential for finding the cures to diseases such as cancer.



Primer Design and Testing The SNP in question is labeled as rs1805008 and is found on the MC1R gene on the 16th chromosome. There is a point mutation at location 89919736 where the cytosine nucleotide has been replaced by a thymine nucleotide in the disease associated allele. Carriers of the this mutation are at risk for Parkinson's disease. To target this gene for examination in a PCR, we need both a forward and reverse primer. Counting 20 nucleotides toward the 5' side of the strand from the affected nucleotide, the forward primer was found to read CAGCATCGTGACCCTGCCGC.

ForwardPrimer.jpg

Beginning on the opposite strand of DNA at location 89919936, 200 nucleotides over, another 20 nucleotides was counted off in the direction of the 3' end of the strand to find the reverse primer CTTGTGGAGCCGGGCGATGC.

ReversePrimer.jpg

When these primers were tested using the UCSC site, a 220 bp result from the 16th chromosome was reported, indicating proper primers had been found for the SNP in question.

Citation for the above images: