BME100 f2016:Group12 W8AM L4

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Group 12, Best Group

Name: Chris Beall
Team Member
Name: Sheigh Cox
Team Member
Name: Joshua Frazier
Team Member
Name: Yanely Valenzuela
Team Member
Name: Kollin Buster
Team Member

PCR LAB

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50µL each: Mix contains Taq DNA Polymerase, MgCl2, and dNTP's
  • DNA/Primer mix, 8 tubes, 50µL 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.
  • Cup for disposable pipette tips
  • Bio-hazard cup for discarded disposable tips
  • Micropipettor
  • Open PCR machine: Shared by two groups

PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G12 + Positive control 58682
G12 - Negative control 54981
G12 1-1 Patient 1, replicate 1
G12 1-2 Patient 1, replicate 2
G12 1-3 Patient 1, replicate 3
G12 2-1 Patient 2, replicate 1
G12 2-2 Patient 2, replicate 2
G12 2-3 Patient 2, replicate 3


DNA Sample Set-up Procedure

  1. First, using the micro pipette, insert 50 μL of DNA/primer mix into an empty PCR tube.
  2. Discard the used pipette tip into designated cup for waste.
  3. Using the micro pipette, insert 50 μL of PCR reaction mix into an empty PCR tube.
  4. Discard used pipette tip into waste cup.
  5. Close and then label the tubes accordingly.
  6. Insert closed tube into thermocycler.
  7. Repeat steps 1-5 for all samples.

OpenPCR program

  • Heated Lid: 100 degree Celsius
  • Initial Step: 95 degree Celsius for 2 mins
  • 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 mins
  • Final Hold: 4 °C






Research and Development

PCR - The Underlying Technology

Function of each component of a PCR reaction:

  • Template DNA: It's the basis for which new DNA strands form. Basically, it's the strand of DNA that's copied to construct mRNA.
  • Primer: Matches the DNA segments needed to copy. It attaches to both the top and bottom DNA segments
  • Taq Polymerase: Copies a cells DNA before it divides in two.
  • Deoxyribonucleotides:A,C,T,G's-- Building blocks that DNA molecules are made of.

What happens to the components during each step of thermal cycling

  • Initial Step: 95 degrees C for 3 minutes: Liquids are coming to temperature to allow the unwinding of the helix.
  • Denature at 95 degrees C for 30 seconds: DNA double helix separates. This creates two single stranded DNA molecules.
  • Anneal at 57 degrees C for 30 seconds: Single stranded DNA molecules naturally attempt to pair up. There are more primer sequences than DNA. This causes the the primers to lock onto the DNA target strands which prevents the DNA from rejoining.
  • Extend at 72 degrees C for 30 seconds: DNA polymerase is activated. When it locates the primer it adds nucleotides onto the DNA strand. This will continue until it reaches the end of the strand then it falls off.
  • Final Step at 72 degrees C for 3 minutes: Allows the DNA polymerase to finish the addition of nucleotides.
  • Final Hold at 4 degrees C: This step stops the process from continuing.

DNA is made up of four types of molecules called nucleotides. Which base anneals to each base?

  • Adenine(A):T
  • Thymine(T):A
  • Cytosine(C):G
  • Guanine(G):C

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

  • Extend: The DNA polymerase is activated adding nucleotides to the DNA strand.
  • Final Step: This allows the process of adding nucleotides to the DNA strand to finish adding the nucleotides.

Pcrrrr.gif

(http://www.contexo.info/DNA_Basics/polymerase_chain_reaction.htm)



SNP Information & Primer Design

Background: About the Disease SNP SNPs or single nucleotide polymorphisms are among the most common type of genetic variation amongst the population of the world. An SNP will generally appear about once every 300 nucleotides in someone's DNA. While most SNPs are harmless, some can be directly tied to a specific disease due to the fact they can alter a gene's function. Scientists are able to use SNPs to act as an indicator that a person has a specific disease, making diagnostics a lot easier. For example, researchers have been able to use SNPs to predict a person's likely response to individual drugs, susceptibility to environmental factors, and even a person's risk of certain genetic diseases. The specific SNP our group is looking at is found in homo sapiens (humans). It is located on the chromosome 4:113367751. This specific SNP is linked to pathogenic factors, more specifically, it is closely linked to cardiac arrhythmia. Sources: https://ghr.nlm.nih.gov/primer/genomicresearch/snp

A short while ago, a test was done to find which SNP's within genes could be related to what caused the Arrhythmia. The study had 273 people, and some of which had died from from cardiac death, and 20 that were revived. The study showed that NOS1AP and KCNQ1 are two of some the SNP's that are related to cardiac arrhythmia, and are the highest risk factored ones too. http://www.sciencedirect.com/science/article/pii/S1547527113011429

Primer Design and Testing The non-disease primer that was created consists of two primers which amplify the DNA. It consists of both a forward and a reverse primer. The non-disease primer is correct because it resulted in 220bp.

Non-disease primer

The diseased primer that was created consists of two primers which alter the amplification of the DNA. It consists of both a forward and a reverse primer. The diseased primer does not work correctly because it shows a result of no match. the "no match" is due to the change in the non-diseased primer from "C" to "A".

Diseased primer