BME100 f2015:Group1 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: Matt Chrest
Role(s)
Name: Lindsey O'Brien
Role(s)
Name: Jacob Aperi
Role(s)
Name: Michael Otis Clyne
Role(s)
Name: Nichole Torgerson
Role(s)
Name: Alarmel Sira
Role(s)

LAB 4 WRITE-UP

Protocol

Materials

  • Lab Coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 μL each:mix contains Taq DNA polymerase, MgCl2 and dNTP's (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/)
  • DNA/primer mix, 8 tubes, 50 μL each: each mix contains a different template DNA. All tubes have the same forward and reverse primer
  • A strip of empty PCR tubes
  • disposable pipette tips: only use each only once. Never reuse disposable pipette tips or samples will be cross contaminated.
  • A cup for discarded tips
  • Micropipetttor
  • Open PCR machine; shared by two groups

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G1 + Positive control none
G1 - Negative control none
G1 1-1 Patient 1, replicate 1 30522
G1 1-2 Patient 1, replicate 2 30522
G1 1-3 Patient 1, replicate 3 30522
G1 2-1 Patient 2, replicate 1 46645
G1 2-2 Patient 2, replicate 2 46645
G1 2-3 Patient 2, replicate 3 46645


DNA Sample Set-up Procedure

  1. Step 1: Using a micropipettor, move 50 μL of DNA/ primer mix into an empty and clean PCR tube
  2. Step 2: Using a micropipettor with a new tip, add 50 μL of PCR reaction mix to the PCR tube
  3. Step 3: Place the PCR tube into the thermal cycler
  4. Step 4: Repeat steps 1-3 for all 8 samples of DNA
  5. Step 5: Program thermal cycler to moderate the temperature of the solution as described below
  6. Step 6: Start thermal cycler


OpenPCR program

  1. Step 1: Set the temperature of the heated lid to 100°C
  2. Step 2: Raise the temperature of the DNA/primer/polymerase solution to 95°C for 2 minutes so that the two strands of DNA can separate (denature)
  3. Step 3: Allow the DNA to denature for 30 seconds at 95°C
  4. Step 4: Allow the DNA to anneal for 30 seconds at 57°C, so that the primers can attach to the ends of the DNA
  5. Step 5: Allow the DNA to extend for 30 seconds at 72°C, whereby Taq DNA polymerase will transcribe to produce double-stranded DNA
  6. Step 6: Repeat steps 3-5 for a total of 25 cycles
  7. Step 7: Lower the temperature of the DNA/primer/polymerase solution to 72°C for 2 minutes
  8. Step 8: Hold the temperature at a final 4°C





Research and Development

PCR - The Underlying Technology
Functions and components: The template DNA is where the PCR process starts. The DNA strand is going to be used as a mold to create specific DNA segments. The DNA will be separated, copied, reformed, in every PCR trial. Primers are the next tool used in the PCR process. Primers are lab made sections of DNA that bind to a specific locations on DNA, in order to start the copying process. These primers are necessary because the DNA Polymerase cannot bind to the DNA strand unless it is paired with the Primer. DNA Polymerase is the actual copier of the DNA strand, connecting the correct opposite nucleotides to the DNA strand. The Nucleotides are the molecules that connect the two strands of DNA together. There are 4 types of nucleotides, and they form two pairs along the entire DNA strand. The variants are labeled as A,T,C,G, where A and T are pairs leaving C and G as pairs. The different nucleotide pairs will be separated in the Template DNA strand, and then replaced by the polymerase and the primers in the copied segment of DNA.

Thermal Cycling: The PCR process from start to finish takes 5 steps to complete. Step one: the separation step, is where the DNA strand is heated to 95°C, allowing for the DNA to Denature and separate into single stranded molecules.The second step is the start of the bonding process, where the Primers bond to the specific DNA strand segments that we want to copy. The temperature is lowered to 57°C for this process, in order to allow the primer to bond. The third step is: the temperature is then raised to 72°C to active the DNA polymerase and allow it to bind to the DNA strand and the primer. The fourth step: At 72°C again,the DNA strand is transcribed, creating our copied segment. The fifth and final step is when new primers are attached to both the copies and the template strand, allowing for the PCR process to repeat and more copies to be created.

Nucleotide Base-Pairing: The four nucleotides are Adenine, Thymine, Cytosine, and Guanine, and work to build base pairs in the DNA strand. Adenine binds to Thymine, and Cytosine binds to Guanine.The base pairs are formed in the 3rd and 4th steps of the PCR process, because in steps 3 and 4, the primers are places on the template strand, and the Polymerase binds to both the primer and the template strand.


File:PCR Process Pictures Group1 Lab4 1030am.docx Images located from:

DNA Learning Center (2010, March 22). Polymerase Chain Reaction (PCR) [Video file]. Retrieved from https://www.youtube.com/watch?v=2KoLnIwoZKU





SNP Information & Primer Design

Background: About the Disease SNP The single nucleotide polymorphism (SNP) disease is rooted in the nucleotides of the DNA itself. SNP is when a single nucleotide (a nucleotide is the monomer of which DNA is composed) is replaced by another nucleotide. For example, SNP is when a cytosine base is replaced by a guanine base. SNP is the most common genetic variation, however most SNP mutations have no affect on the gene expression and the health of the organism. But that does not mean SNP does not lead to diseases. In fact SNP can lead to a whole range of diseases , ranging from autoimmune diseases such as Celiac Disease, to simpler disorders such as glaucoma. The primer given in the lab is a primer for Homo sapiens. The variation occurs on the 16th chromosome, and is associated with the MC1R gene. The Clinical significance of the SNP is that it is pathogenic. The disease associated with this mutation is Parkinson Disease and susceptibility to Clear Renal Cell Carcinoma.


The MC1R (melanocortin 1 receptor) gene functions as the following; G-protein coupled binding receptor, hormone binding, and an alpha melanocyte stimulating hormone receptor. An allele is one of some variant forms of a gene. The base pairs that are disease-associated with this gene are C, T and G. The SNP of this gene is of the numerical position 89919736.


Primer Design and Testing The disease related SNP is located on the top strand of the DNA sequence, which means it is going from 5' to 3' with a 20 base long sequence. The non-disease forward primer was 5'-CGGGCGCGGCGAGCCGTTGC. The second primer was the non-disease reverse primer, which is located exactly 200 bases to the right of where the diseased SNP was located. This numerical position is 89919936. The non-disease reverse primer was 5'-CTTGTGGAGCCGGGCGATGC. Next, a forward and reverse primer for the disease was needed. The Process was similar except the final base on the non-disease forward primer was replaced with the disease related base found in the SNP. The disease forward primer happened to be 5'-CGGGCGCGGCGAGCCGTTGT. Afterwards was the disease reverse primer which ended up being 5'-CTTGTGGAGCCGGGCGATGC. When the primers were checked using the UCSC In-Silico PCR website our data came back with error. The sequence had a 201 bp which is not valid compared to the 220 bp that it should be. This error could come from entering a wrong base in the sequence or possibly not having the correct primer in the system, which in return would cause some of the data to be off. Lastly, the image of the diseased primers came back as not matching. This could be because of some fragments within the sequence.


alt text

This image of the PCR program shows that there was not a match.