BME100 f2017:Group13 W1030 L4

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The Nightmare Before BME

Daniel Beltran
Matt Castile
Catalina Pardo
Brenna Toshner
Nivina Warner

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 micro-liters each: Mix contains Taq DNA polymerase, MgCl2 and dNtp's.
  • DNA/primer mix, 8 tubes 50 micro-liters 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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G13 + Positive control none
G13 - Negative control none
G13 1-1 Patient 1, replicate 1 52396
G13 1-2 Patient 1, replicate 2 52936
G13 1-3 Patient 1, replicate 3 52396
G13 2-1 Patient 2, replicate 1 37028
G13 2-2 Patient 2, replicate 2 37028
G13 2-3 Patient 2, replicate 3 37208


DNA Sample Set-up Procedure

  1. Move the DNA extracted from the patient to a PCR reaction tube.
  2. Add Primer 1 to the PCR reaction tube with the DNA.
  3. Add Primer 2 to the PCR reaction tube with the DNA and Primer 1.
  4. Add nucleotides to the PCR reaction tube with the DNA, Primer 1, and Primer 2 so that there are materials to build complementary DNA fragments during the PCR process.
  5. Add DNA polymerase to the PCR reaction tube to create the DNA fragments.
  6. Place the PCR reaction tube into the thermal cycler.


OpenPCR program
1. HEATED LID: 100°C
2. INITIAL STEP: 95°C for 2 minutes (DNA "unfolds")
3. NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds (Primers bind or anneal to complementary matches on the target DNA sequence. The double helix separates, creating two single-stranded DNA molecules)
Anneal at 57°C for 30 seconds (Single-stranded DNA molecules naturally attempt to pair up but there are more primer sequences than DNA strands in the tube. Primers crowd their way in and lock onto target before strands can rejoin)
Extend at 72°C for 30 seconds (DNA polymerase is activated)
4. FINAL STEP: 72°C for 2 minutes (DNA polymerase locates the primer attached to the single DNA strand and begins to add the complementary nucleotides. This process continues until the polymerase reaches the end of the strand)
5. FINAL HOLD: 4°C
http://learn.genetics.utah.edu/content/labs/pcr/






Research and Development

PCR - The Underlying Technology

PCR Reaction Components and Their Functions

One main component of a PCR reaction is the template DNA. The template DNA is the sample of DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other. Primers, another important component in a PCR reaction, are short pieces of DNA made in a laboratory. In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy. Primers are necessary because DNA polymerase can only attach onto an existing piece of DNA. Taq polymerase, an enzyme that is used to bind to the primer sequences, is used to extend the second strand by adding nucleotides. Nucleotides are composed of deoxyribonucleotides, which are the single base units (A, T, G, and C) that act as building blocks for new DNA strands. http://learn.genetics.utah.edu/content/labs/pcr/

Thermal Cycling Procedure

The initial step of thermal cycling is to heat up the DNA to its boiling point of 95 degrees Celsius for two minutes so that the DNA unfolds. After denaturing the DNA for 30 seconds, the primers either bind or anneal to complementary matches on the target DNA sequence. The double helix will then separate, creating two single-stranded DNA molecules. Once the single-stranded DNA molecules are created, anneal the DNA at 57 degrees Celsius for 30 seconds. The single-stranded DNA molecules will naturally attempt to pair up, however, there are more primer sequences than DNA strands in the tube. Primers will crowd their way in and lock onto target before strands can rejoin. To activate the DNA polymerase, extend the DNA at 72 degrees Celsius for 30 seconds. Finally, the DNA will locate the primer attached to the single DNA strand and begin to add the complementary nucleotides. This process continues until the polymerase reaches the end of the strand. End the procedure by holding the temperature at 4 degrees Celsius. http://learn.genetics.utah.edu/content/labs/pcr/

Base-Pairing

It is during the annealing and extending part of thermal cycling that the base-pairing takes place. Adenine pairs with Thymine (and vice-versa), while Guanine pairs with Cytosine (and vice-versa.) Annealing is when the system is cooled and the target DNA is bound with the short DNA primers. During extension, the Taq polymerase binds to each PCR primer and begins adding in the corresponding nucleotides. https://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Genetics/basepair.html
Group13ecpic.jpg Group13ecpic2.jpg Group13ecpic3.jpg


SNP Information & Primer Design

Background: About the Disease SNP

SNP is defined as a single nucleotide polymorphism, meaning one of the nucleotide bases (A, T, G, or C) has a common variation among individuals who share the same structure of DNA. Nucleotides are compounds linked to phosphate groups and form the basic structural unit of nucleic acids. SNPs occur in the human genome. The variation, rs769452, is found in Homo sapiens, more specifically, in the chromosome 19:44907853. The clinical significance of this SNP variation is that it is pathogenic and is linked to issues such as Alzheimer's, strokes, and subarachnoid hemorrhage. APOE is defined as Apolipoprotein E. and it is the protein that "binds to a specific liver and peripheral cell receptor and is essential for the normal catabolism of triglyceride-rich lipoprotein constituents." It is an integral part of antioxidant activity, cholesterol, protein binding, and other important functions. When a gene has one of two or more alternative forms, derived from mutations, found in the same place on a chromosome, it is considered an allele. In case of the given SNP, the disease-associated allele contains the codon CTG which is then changed into CCG. Its numerical position is 44907853 (base pairs). https://www.ncbi.nlm.nih.gov/ https://www.thoughtco.com/allele-a-genetics-definition-373460

Primer Design and Testing

The non-disease forward primer would be 5'-AGCGGCCAGCGCTGGGAACT-3’. Since every PCR reaction needs two primers, the non-disease reverse primer would be 5’-​CAGGCCCCCCAAGACTTAGC-3’, located in the numerical position 44908053. The disease forward and reverse primers would be 5’-AGCGGCCAGCGCTGGGAACC-3’, and 5’-CAGGCCCCCCAAGACTTAGC-3’​, respectively. Screenshot 2017-for-BME-group-13.png