Name: Aimen Vanood Role(s): Research and development
Name: Brady Falk Role(s): Machine testing
Name: student Role(s)
Name: student Role(s)
Name: student Role(s)
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
(Add image of the full OpenPCR machine here, from the Week 9 exercise. A Polymerase Chain Reaction (PCR) Machine, as shown in the above image, is a machine that is meant to make large quantities of specific Deoxyribose Nucleic Acid (DNA) sequences. The method used is by completing several heating and cooling cycles to unzip DNA strands and isolate the desired DNA strands.
Experimenting With the Connections
When we unplugged the Liquid Crystal Display from the open PCR circuit board, the machine no longer displayed onything on the LCD screen. When we unplugged the white wire that connects the circuit board to 16-tube PCR block, the machine would not produce any heat through the 16-tube PCR block. Anything connected to the circuit board, such as the LCD display or the 16-tube PCR block, will not run when the connection is disrupted.
Test Run
The first test was run on March 5, 2013 using Machine 13. The sample of DNA was put into the PCR machine and all of the results came out as expected. There were no problems while testing.
First, we collected all the materials needed and correctly identified the eight testing tubes
according to the corresponding ID, patient, and replication.
Step 2
Step 3...
PCR Reaction Mix
What is in the PCR reaction mix?
DNA/ primer mix
What is in the DNA/ primer mix?
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
(Add a write-up of the information discussed in Week 3's class)
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
DNA replication is a biochemical process that highlights the importance of making and breaking bonds. DNA is composed of a sugar-phosphate backbone and bases. A phosphate group, a ribose, and a base bonded together create a nucleotide. The chemical attraction between complementary nucleotides results in the double-helix structure of DNA. The base adenine is complementary to thymine, and similarly guanine is complementary to cytosine. The chemical structures of these nucleotides make them ideal for hydrogen bonding to their complementary base pairs. DNA contains the genetic information of a cell, and is unique to every organism. In some cases, a mutation in DNA can cause serious diseases, such as cancer. A gene mutation is typically an error in the complementary base pair rules, an adenine being bonded to a cytosine for example. Polymerase chain reaction, or PCR, is a procedure that is used to detect these mutations early on so action can be taken to prevent the disease before it increases in severity. PCR amplifies small segments of DNA to allow doctors to pinpoint any mutations with more ease. The procedure for this relies heavily on using temperature to either break bonds or encourage binding.
The extracted segment of DNA, called the template DNA, is placed in an aqueous solution also consisting of dNTP’s, or extra bases. The temperature of the mixture is raised to 95°C, causing the hydrogen bonds between bases to break, consequently unzipping the double helix. A primer, which is a short fragment of artificially synthesized DNA, is added once there are two separate strands. Typically, primers consist of 20 base pairs that are complementary to the DNA strands. There are two kinds of primers, a forward primer and a reverse primer. Once they are added, the temperature of the solution is reduced to 57°C, encouraging primer annealing to occur with the template DNA strands. The forward primer binds from left to rich, while the reverse primer binds from right to left. This process results in segments of DNA that are double stranded. In order to create a complete double strand, the temperature is increased again, this time to 72°C. This activates tag polymerase, an enzyme that seeks areas with primers and continues down the DNA strand grabbing base pairs from the surrounding solution and binding them to the template strand. Magnesium chloride is a small molecule that serves as a cofactor for tag polymerase, helping the protein efficiently do it's job. This procedure as a whole is usually repeated 30 times. In this way, a small portion of DNA can be replicated repeatedly.
Polymerase chain reaction can be used in two ways to detect cancer genes. One way is to use a healthy DNA primer, which will consequently not bind to a gene mutation. This will cause one of the DNA strands to not replicate, indicating a problem. The other way is to design a primer so that it binds to the cancer mutation. In this case, if a positive result occurs, it will signify the presence of a cancerous gene. These two methods can be combined as well, in order to make sure the experiment did not fail.
BME 103 Spring 2013
