BME100 f2017:Group9 W1030 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

Name: Samantha Balconi
Name: Cassity Jones
Name: Carlos Ruiz
Name: Rachel Oberlander
Name: Nicholas Lee

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat
  • Disposable gloves
  • PCR reaction mix, 8 tubes, 50μL each: mix contains Taq DNA polymerase, MgCl2, and dNTP's
  • DNA/prer mix, 8 tubes, 50μL each: each mix contains a different template DNA
  • Strip of empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G9 + Positive control none
G9 - Negative control none
G9 1-1 Patient 1, replicate 1 54046
G9 1-2 Patient 1, replicate 2 54046
G9 1-3 Patient 1, replicate 3 54046
G9 2-1 Patient 2, replicate 1 94177
G9 2-2 Patient 2, replicate 2 94177
G9 2-3 Patient 2, replicate 3 94177


DNA Sample Set-up Procedure

  1. Get DNA sample from patients
  2. Put DNA sample into designated PCR tube
  3. Add Primers 1 and 2 into the PCR tube
  4. Add nucleotides to PCR tube
  5. Add DNA polymerase to PCR tube
  6. Place PCR tube into DNA Thermal Cycler
  7. Remove and analyze DNA Sample
  8. Conduct two more trials with same patient DNA
  9. Repeat steps 1-7 for the second patient


OpenPCR program

Heated Lid: 100 CHEATED 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

Components Functions
Template DNA DNA sequence which is to be copied in PCR reaction
Primers Used to target specific portion of DNA wanted to be copied
Taq Polymerase Builds new strand of DNA that was marked by primers
Deoxyribonucleotides (dNTP's) Single units for the bases A, T, C, and G
Steps Event
INITIAL STEP: 95°C for 2 minutes: Heat up the sample
Denature​ at 95°C for 30 seconds: Template DNA split into single strands
Anneal​ at 57°C for 30 seconds: Primers bind to complimentary matches on DNA sequence
Extend​ at 72°C for 30 seconds: Taq Polymerase bind to primers and add nucleotides, extending second strands
FINAL STEP: 72°C for 2 minutes: Keep the extend process to continue
FINAL HOLD: 4°C: Stop the process and stir
Nucleotide Base Pair
Adenine (A) T
Thymine (T) A
Cystosine (C) G
Guanine (G) C

The base-pairing occurs during the anneal and extend steps of the cycle. The primer attaches to a complementary base during the anneal. The extension step pairs all the bases of the DNA strand starting from the primer.



SNP Information & Primer Design

Background: About the Disease SNP

SNP stands for single nucleotide polymorphism. It accounts for the large portion of genetic variation between individuals by differing the nucleotides in certain sections of the DNA. Most of the time, SNPs are used as biological markers and do not have a damaging effect on the individual. However, when the SNPs lie close to or within a gene they are more likely to play a direct role in disease. The specific SNP, rs769452, is found in Homo sapiens on the chromosome 19:44907853. The clinical significance of this SNP is that it is pathogenic and is linked to Alzheimer's Disease. APOE stands for Apolipoprotein E and is the gene where the SNP rs769452 is located. APOE is responsible for carrying cholesterol through the bloodstream. Three unique terms include amyloid-beta binding, antioxidant activity, and cholesterol binding. An allele is the specific location of a gene on a chromosome. The non-disease allele has the codon CTG while the disease allele has the codon CAG. The numerical position of the SNP is 44907853.

Primer Design and Testing

The results of the primer test showed that the sequence of DNA that would be replicated would be what lies in the 5' to 3' direction after the forward primer and would end before the start of the reverse primer.

Primers Group91030.jpg