BME100 s2018:Group4 W1030 L4

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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
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OUR TEAM

Name: Amanda
Tran
Name: Maren
Eltze
Name: Jonathan
Kendall-Jackson
Name: Cesar
Carreto

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mixture, 8 tubes, 50 μL each (this mixture contains Taq DNA Polymerase, dNTP's, and MgCl2
  • DNA/Primer mixture, 8 tubes, 50 μL each (each of these mixtures contains a different template DNA and all of these tubes contains the same forward and reverse primer)
  • A strip of empty PCR tubes
  • Disposable pipette tips (never use more than once)
  • Cup for discarded pipette tips
  • Micropipettor
  • Open PCR machine (shared by two groups)


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G4 + Positive control none
G4 - Negative control none
G4 1-1 Patient 1, replicate 1 38270
G4 1-2 Patient 1, replicate 2 38270
G4 1-3 Patient 1, replicate 3 38270
G4 2-1 Patient 2, replicate 1 31977
G4 2-2 Patient 2, replicate 2 31977
G4 2-3 Patient 2, replicate 3 31977


DNA Sample Set-up Procedure

ThermalCycling G4.png


OpenPCR program


  • Heated Lid: 100ºC
  • Initial Step: 95ºC for 2 minutes
  • Number of Cycles: 25

- Denature at 95ºC for 30 seconds - the DNA helix separates to create two single-stranded molecules of DNA

- Anneal at 57ºC for 30 seconds - the primers attach to the single-stranded molecules of DNA

- Extend at 72ºC for 30 seconds - the DNA polymerase is activated, attaches to primers, and begins to add complementary nucleotides

  • Final Step: 72ºC for 2 minutes
  • Final Hold: 4ºC




Research and Development

PCR - The Underlying Technology

There are four main components of a PCR reaction, which are Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTPs). Template DNA is a sample of DNA used to copy multiples of it. The function of primers are to attach to sites on DNA to the DNA strands that are at either end of the segment one would want to copy. The function of Taq Polymerase is to read the DNA and attach matching nucleotides to create DNA copies. And deoxyribonucleotides (dNTPs) are the building blocks that link to form a strand of DNA.

Thermal cycling is a process in which involves all of the above components. During the initial step, the DNA double helix structure unwinds. Then the DNA splits into 2 single stranded DNA molecules, which is during the denature step at 95 degrees for 30 seconds. Then, Extend at 72 degrees for 30 seconds is where the DNA polymerase binds to the primer and then adds complementary DNA nucleotides to the template strand until polymerase reaches the end and falls off. Then in the final step, the prior 3 steps are repeated to create new cycles and to make additional new DNA strands and cycles that come afterwards will strictly only have the desired DNA fragments. In the final hold, DNA rewinds back to original double helix structure.

DNA is made up of four nucleotides, which are Adenine (A), Thymine (T), Cystosine (C), and Guanine (G).

The first step is during Anneal because the temperature is cool enough that nucleic acids can pair with each other but 57 degrees favors the primers to bind to the template strand of DNA and add the first few base-pairs before each template strand can bind with each other again. Next is Extend at 72 degrees because that this point the temperature is more favorable for base pairing so DNA polymerase binds to the primer and then adds complementary bases to the template strand until completion.



SNP Information & Primer Design

Background: About the Disease SNP

A nucleotide is a compound that is formed of a nucleoside that is binded to a phosphate group, and these nucleotides then become structural units for Deoxyribonucleic acid (DNA) [[1]]. A polymorphism is the variance of genetics within a population, where natural selection can then apply. Therefore, a single nucleotide polymorphism (SNP) is the variance of a single nucleotide or between nucleotides within the genome within a species. The given SNP is rs1044498, which is found in humans (homo sapiens), and this variation is found on chromosome 6. The listed clinical significance of this SNP is pathogenic or rather to demonstrate whether a person is benign or at a risk factor of type 2 diabetes and obesity in relation to insulin resistance[[2]]. The ENPP1 stands for ectonucleotide pyrophosphatase/phosphodiesterase1 [[3]]. The function of ENPP1 protein is to control cell response to the hormone insulin, which plays a role in the regulation of sugar levels throughout the body [[4]].Some other functions include exonuclease activity, insulin receptor binding, and protein homodimerization activity[[5]]. An allele is defined as a variant form of a gene, which arise by mutation and are found at the same place on a chromosome. The non-disease allele contains the codon AAG on the given SNP. A change is this allele, specifically at the A position, is liked to the disease. The disease-associated allele contains the codon CAG [[6]]. The numerical position of this SNP is 131851228.

Primer Design and Testing

Non-disease forward primer (20nt): 5' TTC AGA TGA CTG CAA GGA CA 3'

Non-disease reverse primer (20nt): 5' TGT TTA AAA GTT TCT TTA AT 3'


Disease forward primer (20nt): 5' TTC AGA TGA CTG CAA GGA CC 3'

Disease reverse primer (20nt): 5' TGT TTA AAA GTT TCT TTA AT 3'

UCSC PCR.png UCSC NoMatches.png

The primer test showed that the primers were located on chromosome 6 and were 220 base pairs apart, which is what was found in the primer design as they were found 200 base pairs apart and were both 20 bases long. The disease primers display no match in the results, which makes sense due to those primers not intended to be found along the human genome as it is a part of the SNP mutation.