OUR TEAM

Name: Wade Patrick Machine Engineer

Name: Liann Klein Machine Engineer

Name: Haylee Poncy Protocol Planner

Name: Kyle Labban Protocol Planner

Name: Alexandria Lam R&D Scientist

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

System Design

8 wells were added, this is a good amount to increase sample size significantly yet not to drastic of a change that will affect the machine completely.

The additional wells will also slightly elongate the lid

Key Features
The major change of the PCR machine was that eight extra wells were added to increase the sample size from 16 to 24. With the addition of the extra wells, the machine will be able to run more samples. This also affects the size as the additional wells will cause it to be longer than the original design.

Instructions
The instructions for assembling the PCR machine will mostly stay the same the only difference will be that the additional wells will cause the lid to be longer than the original design.

Protocols

Materials

PCR Protocol

1. Using a pipette, transfer 1.0-1.5 micro liters of the DNA sample into the desired number of test tubes.
2. Pipette approximately 3 micro liters of reagent solution into each of the test tubes with the DNA.
3. Invert tubes, then turn upright to mix solution.
4. Plug in OpenPCR Machine and turn it on.
5. Open lid and place tubes into holder in PCR machine.
6. Close the lid.
7. Add the following cycles on the OpenPCR program:
   1. Stage 1: 1 cycle, 95 degrees Celsius for 3 minutes
   2. Stage 2: 30 cycles, 95 degrees for 30 seconds, 57 degrees for 30 seconds, 72 degrees for 30 second
   3. Stage 3: 72 degrees for 3 minutes
   4. Hold: 4 degrees
8. Run reaction.
9. After the program is complete, open the lid and remove tubes for further analysis.

DNA Measurement Protocol

1. Collect samples generated from "PCR Protocol".
2. Using separate pipettes for each individual sample, transfer the 150μL into the larger test tubes containing 400 mL of the buffer solution.
3. Set up the fluorimeter machinery as instructed, ensuring that the system is devoid of any light, as it may prevent accurate readings. Use a "blank" sample using distilled water to ensure all machinery and processes are in order.
4. Using the fluorimeter equipment, add two drops of each sample onto the glass plate, followed by two drops of SYBR green. When placing the drops, one should ensure that they are initially spaced out, as they will combine when more substrate is added.
5. Close the system down, again preventing any light from entering the system. To record the results, a photo will be used to visually measure the presence of a positive or negative result. The fluorimeter set comes with a stand to enable a SmartPhone to be utilized. Individuals will obtain the most accurate results by setting the ISO at 800 and turning off the flash setting.
6. Using a different pipette for waste products, clear the sample from the glass tray, move the tray forward, and repeat with the next sample. The waste samples can be placed in a separate plastic cup, and eventually disposed of in a biohazard bin.
7. Repeat this process until all samples have been measured and photographed.

Fluorimeter Set Up

Photo origin: http://openwetware.org/wiki/BME103:T130_Group_9

Positive Result

Negative Result

Research and Development

Bayesian statistics

A=Alzheimer's B=Positive test Result

P(A / B) = P(B / A)P(A) / P(B)

Background on Disease Markers

Alzheimer's disease is a form of dementia that occurs with loss of brain function. It affects multiple areas of the brain associated with memory, language, personality, perception, and cognitive skills. The disease typically manifests itself through forgetfulness, but gradually progresses to inability to perform basic functions, speak, and recognize family members. Currently, there is no cure. Treatment tries to slow down the disease or at the least, manage symptoms.

An SNP related to Alzheimer's disease is rs1466662 (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1466662). It is located on chromosome four, the intron region of NM_001142552.1 and arises from a missense mutation replacing an A with a T. It is the most significant SNP outside of the SNP linked to APOE.

Primer Design

The backwards primer is TAT TTT TAG AAG CGA TAA AA. The forwards primer is GCC TCT TTG CCC TCT GTT TT. An allele not containing the disease will not have the sequence that allows the primers to bind. If the primers cannot bind, then that means Taq polymerase does not know where to bind. If Taq polymerase does not bind, then the sequence does not get replicated. Therefore, there will be no PCR product. Conversely, if the disease allele is present, the primers will bind. Taq polymerase will then be able to bind to the DNA and replicate the strands, creating more double-stranded DNA yielding a PCR product.

Illustration

The wanted gene in the figure above refers to rs1466662.

Photo courtesy of http://users.ugent.be/~avierstr/principles/pcr.html