LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
This PCR machine is a laboratory apparatus that is used to take segments of DNA via the polymerase chain reaction (PCR). These machines are used in laboratories to facilitate cycles of reactions that vary in temperature. This device has 16 positions for 16 mini-PCR tubes (or cuvettes) to contain the reaction mixtures. This machine raises and lowers the temperature of the block in pre-programmed steps in order to achieve the different steps of dividing and multiplying strands of DNA.
Components of PCR
The heat block begins the PCR process. This piece of the machine can be precisely depended; it can create a temperature that the machine is programmed for in order to complete an important portion of a PCR cycle.
The OpenPCR features an adjustable-height heated lid, which heats the top of the PCR tubes in order to prevent any form of condensation that may affect the concentration within the PCR tubes.
All PCR machines require a DNA template that consists of a targeted DNA region that is to be replicated and amplified. Two primers are use that correlate to the ends of each of the DNA strands of the DNA target. These machines employ a heat-stable Taq polymerases. These polymerases assemble a new DNA strand from the nucleotides by using single-stranded DNA as a template and DNA primers. These are required for the beginning process of DNA synthesis. dNTPs (deoxynucleotide triphosphates) are the building blocks from which the DNA polymerase synthesizes a new DNA strand. Most PCR machines operate using a method of thermal cycling.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine LED screen did not work properly and was turned off.
When we unplugged the white wire that connects (part 6) to (part 2), the machine's heat plate was disconnected. The LED screen displayed that the temperature was currently -40 degrees Celsius.
Group 14 performed a 12-cycle (approximately 45 minutes) test run on the device on March 19, 2014. The machine passed appropriately and no portions of the device malfunctioned. The OpenPCR machine was easily pre-programmed using the program provided via USB (using the computer). The device will be ready to be used for next week's portion of the lab; an actual PCR experiment will be performed.
Thermal Cycler Program
HEATED LID: 100 degrees Celsius
INITIAL STEP: 95 degrees Celsius for 2 minutes
NUMBER OF CYCLES: 35
Denature at 95 degrees Celsius for 30 seconds,
and Anneal at 57 degrees Celsius for 30 seconds,
and Extend at 72 degrees Celsius for 30 seconds
FINAL STEP: 72 degrees Celsius for 2 minutes
FINAL HOLD: 4 degrees Celsius
Thermal Cycle Steps
During the denaturing step of thermal cycling, the PCR is heated to cause DNA melting of the DNA template, creating two single-strands of DNA.
In the second step, the temperature is lowered and annealed, where the two DNA strands become templates for DNA polymerase to replicate the target DNA. The selectivity of PCR derives from primers that correlate to the DNA region that is targeted for replication and amplification.
During the elongation step, the temperature is increased where the Taq polymerase has its optimum activity level. At this step, a new DNA strand complementary to the DNA template is synthesized by adding dNTPs.
DNA Sample Set-up
The patient's ID's are: 26734 and 22707 for Patient 1 and Patient 2, respectively.
| Positive Control
|| Patient 1, Replicate 1
|| Patient 1, Replicate 2
|| Patient 1, Replicate 3
| disease DNA
|| ID: 26734
|| ID: 26734
|| ID: 26734
| Tube Label: A00
|| Tube Label: A01
|| Tube Label: A02
|| Tube Label: A03
| Negative Control
|| Patient 2, Replicate 1
|| Patient 2, Replicate 2
|| Patient 2, Replicate 3
| non-disease DNA
|| ID: 22707
|| ID: 22707
|| ID: 22707
| Tube Label: B00
|| Tube Label: B01
|| Tube Label: B02
|| Tube Label: B03
DNA Sample Set-up Procedure
- Download the Open PCR software to your computer if needed.
- Plug in the Open PCR machine to a nearby electrical outlet. Afterwards, plug in the Open PCR machine to your computer using the USB 2.0 Cable.
- Locate the "access wall" that is located towards the bottom of the unit. The wall that does not have a slot and is labeled "Open PCR 1 Janowski and Perfetto," is the access wall.
- Carefully remove all screws in the access wall.
- Identify the parts and their functions inside the Open PCR using the Open PCR manual.
- Re-assemble the access wall
- Place empty PCR tubes inside the machine. Close the lid and tighten it so the inner plate touches the tubes.
- Make a TEST RUN by clicking the "more options" button. Enter the appropriate values in a new program. These values consist of the Thermal Cycler Program (found above)
- Start Run. Wait 2 hours.
- After two hours if the machine successfully finished, write the word "PASS" on the lab tape. If not write "FAIL"
- Get patient ID's from the Teaching Assistant.
- Set up 8 total reactions. Label appropriately
PCR Reaction Mix
- What is in the PCR reaction mix?
- Each PCR reaction mix contains Taq DNA polymerase, MgCl2,and dNTP's.
DNA/ primer mix
- What is in the DNA/ primer mix?
- The DNA/primer mix contains a different template of DNA. All tubes have the same forward primer and reverse primer.
Research and Development
PCR - The Underlying Technology
Polymerase Chain Reaction (PCR) is a method of creating multiple copies (2^n; n = number of 3-step cycles) of DNA.
DNA Base Pairing
Attached to each ring are nucleotide bases, organic molecules that serve as the monomers of nucleic acid and the building blocks of nucleic acids. These bases are one of four bases: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). The first two (A, G) are purines and the second two (C, T) are pyrimidines. A base pair is a pair that consists of the matching between two bases: A-T or C-G. Each DNA base pair consists of a purine and a pyrimidine.
After searching rs237035, this variation was found to be related to the Homo Sapiens species. It is located on chromosome 6 and is found to be associated with the SUM04 (small ubiquitin-like modifier 4) and TAB2 genes. These genes are linked to mainly diabetes; however, they have been linked to VKH syndrome and arthritis. No clinical significance was found for this SNP.
SUM04 is found to be attached to proteins and can control a target patient's proteins' sub-cellular localization, stability, or activity.
An allele is one of a number of forms of the same gene or an alternative form of a gene for a character producing different effects. The non-disease allele contains GTG. A change in the "G" position (to ATG) is known to be associated to the disease allele.
The numerical position of the SNP is: 149721690. The disease-associated allele is: AACCACGGGGATTGTCAGTG. As stated before, two primers are needed for a PCR reaction to complete one 3-step cycle; thus, the reverse (complimentary) allele 200 bases to the right of the disease SNP is: AGTTTTCTAATTGAGAATGC.