BME100 f2015:Group7 1030amL4

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png BME 100 Fall 2015 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
BME494 Asu logo.png


OUR TEAM

Name: Haley Archambault
Name: Carson Minarik
Name: Hannah Graff
Name: Courtney Gadbois
Name: Elizabeth Meneses
Name: Thalia Dinh

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

  • 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: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
7 + Positive control none
7 - Negative control none
7 1-1 Patient 1, replicate 1 93258
7 1-2 Patient 1, replicate 2 93258
7 1-3 Patient 1, replicate 3 93258
7 2-1 Patient 2, replicate 1 53331
7 2-2 Patient 2, replicate 2 53331
7 2-3 Patient 2, replicate 3 53331


DNA Sample Set-up Procedure

  1. Step 1: Add extracted DNA into PCR tube
  2. Step 2: Add Primer 1 into PCR tube
  3. Step 3: Add Primer 2 into PCR tube
  4. Step 4: Add nucleotides (A, T, G, C) into PCR tube
  5. Step 5: Add DNA polymerase into PCR tube
  6. Step 6: Place PCR tube into thermal cycler


OpenPCR program

  1. HEATED LID: 100°C
  2. INITIAL STEP: 95°C for 2 minutes
  3. NUMBER OF CYCLES: 25 - Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and
  4. Extend at 72°C for 30 seconds
  5. FINAL STEP: 72°C for 2 minutes
  6. FINAL HOLD: 4°C






Research and Development

PCR - The Underlying Technology
Question 1: What is the function of each component of a PCR reaction?

Template DNA is the original DNA that is split and is used for primers to copy its DNA. Primers are components that attach onto specific sites on DNA strands to helps start off the process of copying the DNA. In order to copy the DNA with the primers, Taq Polymerase comes into play. They act as machines that attach Deoxyribonucleotides (the A's, C's, T's, and G's that make up DNA. They are the building blocks that code DNA that can be turned on and off for different functions) to the template DNA to make DNA copies.

Question 2: What happens to the components (listed above) during each step of thermal cycling? The thermal cycling process occurs by first going through the initial step of 95°C for 3 minutes by having the DNA double helix separating. Denature at 95°C for 30 seconds occurs when the double helix creates two single stranded DNA molecules. During anneal​ at 57°C for 30 seconds the single stranded DNA attempts to pair up but is prevented by the primer. During extend​ at 72°C for 30 seconds, DNA polymerase activates adding nucleotides onto the DNA strands by locating primers. The final step at 72°C for 3 minutes creates an overall result of two double strands of DNA. The final hold at 4°C would be the whole process repeating itself by creating several fragments.

Question 3: The nucleotides Adenine pairs up with Thymine and Cytosine pairs up with Guanine. Base Anneals: Adenine (A): Thymine (T) Thymine (T): Adenine (A) Cytosine (C): Guanine (G) Guanine (G): Cytosine (C)

Question 4: During which two steps of thermal cycling does base-pairing occur? Explain your answers. Thermal cycling does base-pairing in Anneal and Extend. In Anneal the primers are also DNA fragments which have nucleotides that pair up with a certain site on the template DNA. And for Extend, DNA polymerase adds nucleotides onto the DNA stand.




SNP Information & Primer Design

Background: About the Disease SNP A diseased single nucleotide polymorphism is a change in a single nucleotide, which is one of the four bases; Adenine, Cytosine, Guanine, Thymine. This change causes a change in the way polymerase reads and produces the sub sequential proteins. In our lab we were given a strand of DNA from the Homo Sapien species which had a single nucleotide polymorphism on chromosome 16:89919736. This chromosome is relate to the MC1R gene and relates to the disease renal cell carcinoma or a combination of melanoma and Parkinson's.

The MC1R stands for melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor). One of the functions of MC1R is g-protein couple with peptide receptor activity. Another function is MC1R binds proteins. The third function is MC1R has melonocmratin receptor activity.

An allele is a form of a gene that is found through mutation and found in the same position as the original gene. In the gene the allele is mutated seeing a change in the original code from CGG to TGG

Position:89919736

Diseased:

5'-CAGCATCGTGACCCTGCCGT


Non-Diseased Forward Primer:

5'-CAGCATCGTGACCCTGCCGC


Non-Disease Reverse Primer:

Position:89919936

5'-GGCATCGCCCGGCTCCACAA


Disease Forward Primer: 5' CAGCATCGTGACCCTGCCGT

5' GTCGTAGCACTGGGACGGCA

Primer Design and Testing When i tested the non-disease primers on the website my search results did indeed reveal the same 220 bp sequence in the fourth chromosome. Then when i put in the disease primers the resulting search yielded no matches. This is probably because the disease has not been seen in the been mapped in the Human Genome because it was eradicated before the project started and was therefore unmapped.