BME100 s2018:Group5 W0800 L4

From OpenWetWare
Jump to navigationJump to search
Owwnotebook icon.png BME 100 Spring 2018 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: Priya Borah
Role(s)
Name: James Crichton
Role(s)
Name: Andrew Holder
Role(s)
Name: Kenzie Tommaney
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
  • 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
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G5 + Positive control none
G5 - Negative control none
G5 1-1 Patient 1, replicate 1 70416
G5 1-2 Patient 1, replicate 2 70416
G5 1-3 Patient 1, replicate 3 70416
G5 2-1 Patient 2, replicate 1 90490
G5 2-2 Patient 2, replicate 2 90490
G5 2-3 Patient 2, replicate 3 90490


DNA Sample Set-up Procedure
Step 1: Add the desired DNA samples from the patients to the PCR test tube
Step 2: Add the first primer to the PCR tube (using a different pipet tip)
Step 3: Add the second primer to the PCR tube (using a different pipet tip)
Step 4: Add the dNTP, such as the dATP, dTTP, dGTP, and dCTP which are the nucleotides (using a different pipet tip)
Step 5: Add the DNA polymerase to the test tube (using a different pipet tip)
Step 6: Repeat this process as desired for all of the DNA samples that are required
Step 7: Place the test tube(s) into the thermal cycler to bein the process

OpenPCR program

  • Heated Lid: 100°C
  • Initial Step: 95°C for 2 minutes
  • Number of Cycles: 25
  • Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and extend at 72°C for 30 seconds
  • Final Step: 72°C for 2 minutes
  • Final Hold: 4°C






Research and Development

PCR - The Underlying Technology
The blueprint to everything in the world is DNA, and many advances in the world today are based on the analysis, interpretation, and modification of DNA. Polymerase Chain Reaction (PCR) is a method that makes the analysis of DNA possible and easier. For PCR, there are specific parts of DNA are needed to test, so the DNA samples are samples that the process will choose to replicate. The primers and the DNA polymerase that are used for this reaction, are used to get the DNA split apart in the right spots and the nucleotides in the correct areas. The nucleotides that are used and entered this reaction are needed to match up and provide corresponding base pairs in the reaction. The thermal cycling that is necessary for this reaction because the temperature that is desired (95 degrees Celsius) helps the DNA go from the double helix to the desired linear shape. With temperatures in the cycle used and necessary in the reaction (57 degrees Celsius for the primers and 72 degrees Celsius for the polymerase), the reaction will go and continue to have the desired amount of DNA.

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

In a PCR or a Polymerase Chain Reaction, there are 4 main components that include the template DNA, primers, taq polymerase and deoxyribonucleotides or dNTP's. The DNA template is the DNA that we want to replicate using the PCR reaction. It is usually placed within a PCR tube and kept cool before being placed in the thermocycler. The primers, usually two used in PCR are the components that serve as the starting point of the DNA replication before taq polymerase can come in and replicate the DNA. Taq polymerase is the enzyme that is responsible for copying and creating the DNA from the template strand in order to create a new identical strand of DNA. The dNTP's are the nucleotide bases that will make up the new copy of the DNA strand. For instance the dNTP's include dATP, dTTP, dCTP and dGTP.

What happens to the components during each step of thermal cycling?

Thermal cycling is one of the main processes of the PCR reaction. It includes 6 steps that requires the DNA in the PCR tube to be heated at 95°C for 2 minutes, denatured at 95°C for 30 seconds, annealed at 57°C for 30 seconds, extended at 72°C for 30 seconds, kept at 72°C for 2 minutes and then finally held at 4°C. In the initial step all of the materials including the template, two primers, taq polymerase and dNTP's are heated from room temperature to 95°C. At this point the template strand is denatured into two strands of DNA molecules. Next, in the annealing step the temperature drops to 57°C so that the two primers can each attach to the two single strands of DNA. Then in the the extending step of the thermal cycling, the temperature goes back up to 72°C in order for the taq polymerase to attach to the two primers and attach dNTP's to the single template strands of DNA.

ATGC

DNA is made up of four types of nucleotides designated by the letters A, T, C and G. Adenine and guanine are classified as purines while cytosine and thymine are classified as pyrimidines. These classifications are due to the structure of the nitrogen bases and phosphate groups of the nucleotides. Adenine and thymine are two bases that pair together and cytosine and guanine are another pair. Hydrogen bonding is the reason that these base pairs the purines and pyrimidines can stick together to form DNA.

During which two steps of thermal cycling does base pairing occur?

During thermal cycling the two steps where base pairing occur are during annealing and extending. The temperature drops to 57°C which is what allows the two primers to attach themselves to the DNA strand.

Group5 W0800 L4 Illustration.png

SNP Information & Primer Design

Background: About the Disease SNP

For this lab, we will be investigating a disease single nucleotide polymorphism, or SNP. A nucleotide is a basic building block of DNA or RNA made from a base, sugar and phosphoric acid. The base for DNA can include adenine, thymine, guanine, and cytosine and these are placed in order to create a DNA sequence. When a nucleotide in a genome sequence is changed, it can create a polymorphism, or a genetic variation in the DNA of a population that is relatively common.

The SNP we are testing for, rs1044498, is found in Homo sapiens and is located in the 6:131851228 chromosome. For clinical significance, it is considered benign. However, it could be a risk factor for obesity, diabetes mellitus type 2, arterial calcification of infancy, susceptibility to insulin resistance, and hypophosphatemic rickets. The SNP was closely associated with the bone disorders osteoporosis and osteopenia, especially when the patient has diabetes. It is recommended to check for this variation in patients with type 2 diabetes to predict these bone disorders.

This variation is found in the DNA sequence ENPP1, which stands for ectonucleotide pyrophosphatase/phosphodiesterase 1. Its main functions are 3’-phosphoadenosine 5’-phosphosulfate binding, ATP binding, and NADH pyrophosphatase activity. The disease SNP forms two alleles, which are one of several versions of a gene, which arise due to mutation at a specific spot on the chromosome. The non-disease allele contains the codon AAG, while the disease-associated allele contains the codon CAG. The numerical position of the SNP is 131851228.

Primer Design and Testing

The non-disease forward primer is 5’-TTCAGATGACTGCAAGGACA-3’

The other primer will be 200 bases to the right of the SNP, so at position 131851428.

The non-disease reverse primer is 5’-TGTTTAAAAGTTTCTTTAAT-3’

The disease forward primer is 5’-TTCAGATGACTGCAAGGACC-3’

The disease reverse primer is 5’-TGTTTAAAAGTTTCTTTAAT-3’


Test results for non-disease primers:

Group5 W0800 L4 nondisease primer.png

Test results for disease primers:

Group5 W0800 L4 disease primer.png

These results match what was expected. The disease primers yielded no results because the website used a non-disease human genome sequence.