BME100 f2017:Group7 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

Sarah Brady
River Rodriquez
Kayla Culhane
Adriane Cana
Sevy Perez

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 primer and reverse primer
  • A strip of empty PCR tubes
  • Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine: shared by two groups
Tube Label PCR Reaction Sample Patient ID
G7 + Positive control none
G7 - Negative control none
G7 1-1 Patient 1, replicate 1 51980
G7 1-2 Patient 1, replicate 2 51980
G7 1-3 Patient 1, replicate 3 51980
G7 2-1 Patient 2, replicate 1 45751
G7 2-2 Patient 2, replicate 2 45751
G7 2-3 Patient 2, replicate 3 45751

DNA Sample Set-up Procedure

  1. Don proper PPE including lab coat and gloves and prepare clean lab bench.
  2. Collect PCR reaction mix, DNA/primer mix, a strip of empty PCR, disposable pipette tips, a cup for discarded tips, and a micropipettor.
  3. Label six empty PCR tubes with patient 1, replicate 1-3 information and patient 2, replicate 1-3 information.
  4. Label two empty PCR tubes with positive and negative control information.
  5. Vortex or mix a PCR reaction mix tube and spin it in a centrifuge.
  6. Prepare a container of ice and place PCR reaction mix and DNA/primer mix tubes on top of it.
  7. Prepare another container of ice and place eight empty labeled PCR test tubes on top of it.
  8. Attach pipette tip to micropipettor.
  9. Pipette 50 μL of DNA/primer mix for appropriate control or patient ID into one test tube.
  10. Dispose of pipette tip in proper container.
  11. Attach pipette tip to micropipettor.
  12. Pipette 50 μL of PCR reaction mix into the same test tube.
  13. Dispose of pipette tip in proper container.
  14. Repeat steps 8 through 13 for all 8 PCR test tubes.
  15. Place labeled PCR test tubes in a DNA thermal cycler.

OpenPCR program

  • On the heated lid, set for 100°C.
  • First step is to heat at 95°C for 2 minutes.
  • 25 cycles must be completed.
  • Sample should be denatured at 95°C for 30 seconds, annealed at 57°C for 30 seconds, and then extended at 72°C for 30 seconds.
  • The last step is to have the sample at 72°C for 2 minutes.
  • The final hold temperature is 4°C.

Research and Development

PCR - The Underlying Technology

Functions of Components in a PCR Reaction

There are four components in a PCR reaction, which include template​ ​DNA​, primers, Taq​ ​Polymerase, and Deoxyribonucleotides (dNTP’s)​. The function of template DNA is to be the subject sample that is analyzed for the experiment and later replicates. The function of primers 1 and 2 is to attach at the opposite ends of split DNA fragments and provide a location for DNA polymerase to later bind. The function of Taq​ ​Polymerase is to withstand high temperatures, bind to the primer sites, and collect free floating nucleotides to attach to the DNA until the strand is complete with base pairs. The function of Deoxyribonucleotides (dNTP’s)​ is to create DNA copies through base pairing from the Taq Polymerase.

PCR Steps

DNA strand before heating at 95°C
There are six steps involved in PCR. During the initial step, the PCR tube is heated at 95°C for 2 minutes. This very high heat separates the DNA into distinct, single strand molecules. The second step corresponds to the first, where the DNA denatures at 95°C for 30 seconds. This continues the process from the first step to allow the DNA to fully separate before the next steps begin.
Annealing at 57°C when primers connect to denatured DNA
During the third step, anneal​ing occurs at 57°C for 30 seconds. At this temperature, primers connect to opposite ends of the denatured DNA molecules, which prevents the DNA strands from rejoining.
Taq polymerase binding to primer at 72°C
During the fourth step, the mixture extends​ at 72°C for 30 seconds. At this point, Taq polymerase becomes active and collects free floating nucleotides to attach to the DNA. Taq polymerase binds to a primer and attaches nucleotides along the strand until it reaches the end of that strand.
Attached nucleotides on DNA strand
The Taq polymerase then falls off. During the fifth and 'final' step, the process of Taq polymerase continues at 72°C for 2 minutes and ensures that all Taq polymerase is synthesized fully. During the sixth step, the mixture is kept at a final hold of 4°C. This is the storage step when the PCR tubes are cooled for storage.

Base Pairing

Base pairing occurs during the steps to anneal​ at 57°C for 30 seconds and extend at 72°C for 30 seconds. During annealing, primer attaches to a DNA strand through base-pairing. During extending at 72°C, DNA polymerase becomes activated and grabs nucleotides from the PCR mixture to attach at a strand specified by the primer. Bases that anneal to each other are as follows, Adenine (A) to Thymine (T) and Guanine (G) to Cytosine (C).


SNP Information & Primer Design

Background: About the Disease SNP

In this part of the lab, the NCBI database was used to find a disease-associated sequence. Key terms related to this search are nucleotides and polymorphisms, as the database enables researchers to locate short genetic variations. Nucleotides are the backbone of nucleic acids, consisting of phosphate connected to sugar connected to a base pair. A polymorphism is a genetic variation found in DNA that usually will divide the population of a species into two or more different phenotypes of the species. The specific single nucleotide polymorphism investigated in this lab was a variation found in chromosome 19:44907853 of the Homo sapiens species. The clinical significance of this SNP is that it is pathogenic and is associated with both Alzheimer’s Disease and stroke. To analyze this SNP, the DNA sequence of the SNP and the surrounding sequence were assessed. The gene corresponding to this SNP is apolipoprotein E (APOE), which has many functions including amyloid-beta binding, antioxidant activity, and cholesterol binding. The disease-associated allele of this gene is CCG. An allele is the different form a gene can have at the same place on a chromosome. It is a variation that occurs through mutations.

Primer Design and Testing

For the primer test, a non-disease forward primer was first designed for rs769452​ based on the numerical position of the SNP (44907853). The forward primer is 5’-AGCGGCCAGCGCTGGGAACT-3’. To design the reverse primer, the reverse strand of the sequence was recorded 200 bases to the right of the disease (44908053). The reverse non-disease primer is 5’-​CAGGCCCCCCAAGACTTAGC-3’. Disease SNP-specific forward and reverse primers were designed by substituting the final base of non-disease forward primer. This makes it the same as the disease SNP nucleotide. The disease reverse primer is 5’-​AGCGGCCAGCGCTGGGAACC-3’ and the disease reverse primer is 5’-​​​CAGGCCCCCCAAGACTTAGC-3’.

The primers were tested by inputting them into the UCSC In-Silico​ ​PCR​ website. The non-disease primers search result showed a 220 bp sequence from the chromosome 19:44907853 (chr19:44907834 through 44908053), which signaled that the primers do in fact work. The submission with disease-specific primers resulted in no match, which signaled that the disease-specific primers were correctly designed. There was no match because the changed allele doesn’t occur in a non-diseased genome, which is what this database uses as a reference.

Test of Non-Disease Primers
Test of Disease Primers