Name: Coley White
Role(s): Protocol Planner
Name: Aimen Vanood
Role(s): Research and development
Name: Brady Falk
Role(s): Machine testing
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
A Polymerase Chain Reaction (PCR) Machine, as shown in the above image, is a machine that is meant to produce 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.
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.
Thermal Cycler Program
Heated Lid: 110°C
Initial Step: temp: 95°C time: 180 sec
Number of Cycles: 35
Denaturing: temp: 95°C time: 30 sec
Annealing: temp: 57°C time: 30 sec
Extending: temp: 72°C time: 30 sec
Final Hold: temp: 4°C
After the thermal cycler program is set up, we then proceeded with the experiment by saving the settings and selecting the "Plug in OpenPCR to Start" option.
DNA Sample Set-up
cancer DNA template Tube label: +
Tube label A1
Tube label A2
Tube label A3
cancer DNA template Tube label: -
Tube label B1
Tube label B2
Tube label B3
DNA Sample Set-up Procedure
- Step 1: First, we collected all the materials and correctly identified the eight testing tubes, with a marker, according to the corresponding ID, patient, and replication.
- Step 2: Then we transferred 50 μL of the given PCR reaction mix into the 8 tubes using a micropipette. It is imperative that new tips are used each time and then properly disposed of to avoid contamination. Also the container of the tips must remain shut when it is not in used so that airborne contamination is avoided as well.
- Step 3: Next, we transferred 50 μL of the DNA/ primer mix into the 8 tubes, but unlike the reaction mix, the DNA must be carefully transferred into the correct tubes. The first four tubes would have one tube that represented the positive control and the next three would contain the DNA of patient 1. For the remaining tubes, one will be used as the negative control and the rest of the three tubes will contain the DNA of patient 2.
- Step 4: Then, the mixtures will need to be placed into the machine, which must be programmed to the correct temperatures and cycles.
PCR Reaction Mix
The mix is composed of:
* Taq DNA polymerase
DNA/ primer mix
The DNA/ primer mix is composed of:
* Either one of the DNA of patient 1 or patient 2.
* A forward and reverse primer, which will be the same and present in all tubes.
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.