BME100 f2015:Group7 8amL4

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BME 100 Fall 2015 Home
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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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Contents

OUR TEAM

Name: Jeremy Atkinson Role: Chief Playmaker, general information, editing
Name: Jeremy Atkinson
Role: Chief Playmaker, general information, editing
Name: Tristan Loveday Role: VP of Operations, general information, SNP info paragraph
Name: Tristan Loveday
Role: VP of Operations, general information, SNP info paragraph
Name: Rafael Lopez Role: Tech Wizard, depiction of SNP process
Name: Rafael Lopez
Role: Tech Wizard, depiction of SNP process
Name: Kelly Harper Role: Token Minority, backup
Name: Kelly Harper
Role: Token Minority, backup
Name: Farah Alyasari Role: Female representative, R&D paragraph
Name: Farah Alyasari
Role: Female representative, R&D paragraph
Name: Andrew Medina  Role: Head Cheerleader, raw information retrieval
Name: Andrew Medina
Role: Head Cheerleader, raw information retrieval

LAB 4 WRITE-UP

Protocol

Materials

  • Lab coat and disposable gloves
  • 8 50 μL tubes of PCR reaction mix (contains Taq, DNA, primers, dNTPs, MgCl2)
  • 8 50 μL tubes of DNA and primer mix (each tube has different DNA but the same primers)
  • Empty PCR tubes
  • Disposable pipette tips
  • Cup for discarded tips
  • Micropipettor
  • OpenPCR machine


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G7 + Positive control none
G7 - Negative control none
G7 1-1 Patient 1, replicate 1 54502
G7 1-2 Patient 1, replicate 2 54502
G7 1-3 Patient 1, replicate 3 54502
G7 2-1 Patient 2, replicate 1 84174
G7 2-2 Patient 2, replicate 2 84174
G7 2-3 Patient 2, replicate 3 84174


DNA Sample Set-up Procedure

  1. Separate and label PCR tubes
  2. Pipette 50μl of PCR mix into each PCR tube
  3. Using separate tips for each sample, pipette the 50μl of each DNA/primer mix into the appropriate tubes
  4. Close tubes securely
  5. Place tubes in thermal cycler

OpenPCR program

  1. Heat lid to 100°C
  2. Heat samples to 95°C for two minutes (initial denaturing)
  3. 95°C for 30 sec (denaturing), 57°C for 30 sec (annealing), 72°C for 30 sec (extension) X25 cycles
  4. 72°C for two minutes (final extension)
  5. 4°C hold until samples are removed



Research and Development

PCR - The Underlying Technology
Image:Cookiebug1.png Image:Cookiebug22.png Image:Cookiebug3344.png

Example of PCR image:pcrakjfadhjklh.jpg



SNP Information & Primer Design

Background: About the Disease SNP

Nucleotides are the basic building blocks of RNA and DNA, and a single nucleotide polymorphism (SNP) is when a single nucleotide in a sequence is different from another sequence. Alleles are different versions of a gene, and an SNP could result in a different allele for a gene. Humans inherit two versions of every gene. The specific variation described by rs1805008 is an SNP found in Homo sapiens. The SNP is located at position 89919736 on chromosome 16. The SNP is clinically significant because it is pathogenic. Rs1805008 is associated with the Melanocortin 1 Receptor (MC1R) gene. MC1R is G protein coupled and binds to hormones known as melanocortins. MC1R regulates mammalian skin color. The SNP in the MC1R gene is associated with skin cancer.


Primer Design and Testing


The SNP rs1805008 is located at position 89919736 on chromosome 16. In order to make a non-disease forward primer, the twenty nucleotide sequence ending in the SNP (reading from the 5’ to the 3’ end of the top strand) was taken. For the non-disease reverse primer, the twenty nucleotide sequence at the location 200 base pairs to the right of the SNP was taken (reading from the 5’ to the 3’ of the bottom strand). For the disease forward primer, the same sequence was used except thymine was substituted for cytosine in the last base pair in accordance with the SNP. The reverse primer was the same for disease and non-disease. The sequences were found using the NCBI page showing the location of rs1805008 on chromosome 16. The primers were checked on the UCSC In-Silico PCR website.


Non-Disease Primer Test Image:NonDiseasePrimerTest.jpeg

Disease Primer Test Image:DiseasePrimerTest.jpg

The non-disease primers are correct because they will resulting in the synthesis of a 220 nucleotide sequence of DNA when they bind to the wild type sequence. The disease primers are correct because they will not bind to the wild-type sequence of DNA (they are designed to amplify the sequence with the SNP), and thus no sequence is found.

NCBI Sequence Image:NCBIsequence.jpg


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