BME100 f2017:Group10 W0800 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Austin Copps
Jacob Morris
Whitney Hirano
Nicole Van Alstine
Osvaldo Pagan

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR Reaction Mix, 8 tubes, 50 microliters each: Mix contains Taq DNA polymerase, MgCl2 and dNTP's
  • DNA/ primer mix, 8 tubes, 50 microliters: 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: use each only once
  • Cup for discarded tips
  • Micropipettor
  • Open PCR machine: shared by two groups

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G# + Positive control none
G# - Negative control none
G# 1-1 Patient 1, replicate 1 76322
G# 1-2 Patient 1, replicate 2 76322
G# 1-3 Patient 1, replicate 3 76322
G# 2-1 Patient 2, replicate 1 12480
G# 2-2 Patient 2, replicate 2 12480
G# 2-3 Patient 2, replicate 3 12480


DNA Sample Set-up Procedure

  1. Step 1: Obtain a sample of DNA,
  2. Step 2: Set up PCR mix containing Taq DNA Polymerase, MgCl2, and dnTP' and place 50 microliters into the tube.
  3. Step 3: Transfer 50 microliters of the DNA/primer solution into same tube


OpenPCR program

Heated Lid: 100 degrees Celsius

Initial Step: 95 degrees Celcius for two min.

Number of Cycles = 25

  • Denature at 95 degrees celsius for 30 seconds
  • Anneal at 57 degrees celsius for 30 seconds
  • Extend at 72 degrees celsius for 30 seconds.

Final Step: 72 degrees Celcius for two min

Final Hold: 4 celcius



Research and Development

PCR - The Underlying Technology

Q1. What is the function of each component of a PCR reaction?

Template DNA: acts as a guide strand that is copied during replication

Primers: attach to sites on the DNA strands that are at either end of the segment you want to copy. They are powerful tools for copying very specific DNA sequences since there is almost no chance they will target the wrong sites.

Taq Polymerase: enzyme (protein) that assembles nucleotides into new strands of DNA

Deoxyribonucleotides (dNTP’s)​: is the string of DNA that consists of a nitrogenous base, a deoxyribose sugar and one phosphate group.

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

  • INITIAL STEP: 95°C for 2 minutes: During this step, high heat causes the template strand to begin to unwind and separate.
  • Denature at 95°C for 30 seconds: Denaturation due to heat causes the DNA to alter its shape by separating completely into single strands.
  • Anneal at 57°C for 30 seconds: Primers bind to the single strands of DNA.
  • Extend at 72°C for 30 seconds: Taq Polymerase binds to the primer template DNA complex that is formed and adds matching base pairs for the DNA.
  • FINAL STEP: 72°C for 2 minutes: Taq polymerase continues to make copies and DNA is fully extended.
  • FINAL HOLD: 4°C: This low temperature allows the DNA to cool in order to become stable.

Q3. DNA is made up of four types of molecules called nucleotides, designated as A, T, C and G

Adenine (A): Thymine

Thymine (T): Adenine

Cytosine (C): Guanine

Guanine (G): Cytosine

Q4. During which two steps of thermal cycling does base-pairing occur? Explain your answers.

During the two steps of thermal cycling, base pairing will occur between the annealing and extension steps. In the annealing step, the DNA is cooled down and the DNA primers bind to the target DNA. After this step, the extension step occurs and the temperature is increased, allowing Taq polymerase to match base primers with the corresponding pair. From this point, the primer pairs are extended and new nucleotide strands are made from the specific DNA sample and are made into numerous copies after each cycle.

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



SNP Information & Primer Design

Background: About the Disease SNP

Single nucleotide polymorphisms are a type of genetic variation that represent a difference in a single DNA building block called a nucleotide. These commonly occur about once every 300 nucleotides and can act as biological markers. When an SNP occurs in a gene or in the regulatory region near the gene, they may affect the gene's function and play a role in disease. Researchers have found that SNPs are useful for predicting a person's risk for developing certain diseases, responses to a certain drug or environmental toxin, and other phenotypic characteristics. An allele is one or two more alternative forms of a gene that arise bu mutation and are found at the same place on a chromosome.


Description of image


Primer Design and Testing

This species is a variation found in Homo Sapiens. The chromosome that the variation is located on is 19:44907853. The clinical significance is pathogenic. Alzheimers disease and strokes are the conditions associated with this SNP. The disease-associated allele contains the codon CCG. The numerical position for SNP is 44907853.

Part 2: APOE stands for apolipoprotein E The APOE function is that it is cause for lipid binding, protein binding, as well as antioxidant activity. An allele is an alternative form of a gene that can arise through mutations and occurs at a specific location on a chromosome (encyclopaedia britanicca) The disease-associated allele of APOE contains the codon CCG.

Part 3:

  • Non disease forward primer- 5’ A G C G G C C A G C G C T G G G A A C T
  • 44908053
  • Non-disease​ ​reverse​ ​primer​ ​(20​ ​nt)​: 5’- C A G G C C C C C C A A G A C T T A G C
  • Disease​ ​forward​ ​primer​ ​(20​ ​nt): 5’-​ ​A G C G G C C A G C G C T G G G A A C C
  • Already validated both non-disease and disease primers.

Sources

Authors of Encyclopaedia Britannica"Allele" Encyclopaedia Britannica. Retrieved from https://www.britannica.com/science/allele. (Accessed on Ocotber 24, 2017). .