BME100 f2016:Group5 W8AM L4

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Contents

OUR TEAM

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LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • PCR reaction mix, 8 tubes, 50 µL each: Mix contains Taq DNA polymerase, MgCl​2​, and dNTP’s (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorl ess-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


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G5 + Positive control none
G5 - Negative control none
G5 1-1 Patient 1, replicate 1 63747
G5 1-2 Patient 1, replicate 2 63747
G5 1-3 Patient 1, replicate 3 63747
G5 2-1 Patient 2, replicate 1 53529
G5 2-2 Patient 2, replicate 2 53529
G5 2-3 Patient 2, replicate 3 53529


DNA Sample Set-up Procedure

  1. Extract DNA into a PCR tube
  2. Add primers to the PCR tube
  3. Add nucleotides to the PCR tube
  4. Add DNA polymerase to the PCR tube
  5. Place PCR tube into the DNA thermocycler
  6. Heat to 95C
  7. Cool to 50C
  8. Heat to 75C
  9. Repeat steps 6-8


OpenPCR program

HEATED 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

Function of PCR DNA template is the DNA that is being copied for replication. Primers are specifically designed to attach to the DNA and will bind to the DNA polymerase. The Taq polymerase adds the corresponding Deoxyribonucleotide to the DNA. The dNTP's are the components that compose DNA.

Thermo Cycling When heated to 95C in the initial and denature steo, the DNA will denature by untwisting and splitting into to separate strands. At 57C in the anneal step, the primers attach to the separate strands. At 72C in the extend and final step, the taq polymerase will bind to the primer and add the corresponding nucleotides to the DNA. At 4C in the hold phase, the DNA strands will return to their double helix shape.

Base Pairing Adenine and Thymine pair together while Guanine and Cytosine pair together.

Cycle Base Pairing When the thermos cycler is at 72C, the Extend and Final step, the polymerase is bonded to the primer on the DNA strand which allows the polymerase to match the nucleotides to their counterparts.



SNP Information & Primer Design

Nucleotides are the monomers that compose nucleic acids. Polymorphism is when there are two or more different forms of a subject that belong to the same habitat.

Background: About the Disease SNP

SNP rs35530544 specie is a homo sapien with a variation located on chromosome 4:113367751. The clinical significance is that the DNA variation was shown to be pathogenic. This particular SSNP is associated with a cardiac arrhythmia syndrome caused by loss of ankyrin-B function. ANK2 stands for Ankyrin-B. ANK2 functions include ATPase binding, cytoskeletal adaptor activity, and enzyme binding. An allele is the different way a gene can be formed by mutation on a chromosome. The non-disease codon of CTC is changed into the disease codon of ATC. The position is 113367751.

non-disease forward primer 5' GGACAGCTCAGCAACAGCAC 3'

The non-disease reverse primer is at 113367951.

non-disease reverse primer 5' TAAAAAGTATTTAAAAACTA 3'

disease forward primer (SNP) 5' GGACAGCTCAGCAACAGCAA 3'

disease reverse primer 5' TAAAAAGTATTTAAAAACTA 3'

Primer Design and Testing




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