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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
(Please finish by 11/7/2012)
Initial Machine Testing
The Original Design
Experimenting With the Connections
When we unplugged the mounting plate from the circuit board, the machine the LCD light and the menu on the PCR machine shut off.
When we unplugged the white wire that connects the circuit board to PCR block that holds the samples, the temperature on the menu on the PCR machine dropped from room temperature to -40.0 degrees Celsius. The white wire is the temperature sensor wire.
Polymerase Chain Reaction
The Polymerase Chain Reaction works by amplifying DNA through the use of a PCR machine and therefore producing a multitude of copies of DNA sequences. These copies can then be further studied to diagnose diseases that are hereditary and infectious, such as cancer and HIV.
After primer is added to the solution, the DNA is then cooled down to 57 degrees Celsius so that the said primer can attach to a template sequence to form a forward primer. The DNA is then once more heated to 72 degrees Celsius so that the replication process can be completed.
(Add your work from Week 3, Part 2 here)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
What is the function of each component of a PCR reaction?
Template DNA: A double-stranded segment of DNA that encodes either a cancerous gene or a normal gene
Primers: Short segments of DNA that bind to a specific sequence of nucleotides (binds to cancer gene)
Taq Polymerase: A protein that serves as the catalyst for the DNA replication; grabs extra nucleotides within the solution and binds them to the "unzipped" strands
Magnesium Chloride: A cofactor that binds to the Taq Polymerase and affects the speed of the reaction; positive correlation between amount of magnesium chloride and reaction speed
dNTP's: Deoxynucleotide triphosphates; extra nucleotide bases in solution that are able to be grabbed and synthesized by Taq Polymerase to replicate DNA strands beyond the primer sequence
• At 95° Celsius: DNA melts and "unzips" to create two one-stranded strips, primers are added to the solution
• At 57°Celsius: Primers attach to the corresponding template sequence they complement, forming one forward primer and one reverse primer
• At 72° Celsius: Taq Polymerase finishes the replication process with the use of dNTP's and magnesium chloride
Why does a cancer gene produce a positive result while a normal gene produces a negative?
• Because the cancer gene has the specific sequence of nucleotides that the primers can bond to, the process can continue and the DNA can be replicated; however, since the normal gene does not include that specific sequence, the primers can never bond to the strands and the process cannot take place.
In order to achieve accuracy of the amplification process as an actual determinant for cancer, Bayes' Rule must be used. This will compute the probability of true positives in coordination with false positives and false negatives to give a realistic prediction for how reliable the PCR process is in detecting the true cancer patients.
Image Credit to OpenPCR.org/use-it/