The OpenPCR machine is a accurate thermocycler that can be used for the dectection and sequencing of DNA. It changes tempuratures quickly for certain sets of time intervals. During these certain interals of time, the DNA's tempurature is first raised to near bolining, creating the DNA to denature into two single strands. After the tempurature is decreased the primers attach to the strands to bracket the target sequence for copying. The tempurature is then increased slightly and the Taq polymerase adds neucleotides to each stand. After this is done the cycles keep repeating until the amplification process is complete. The OpenPCR is used for many things such as human diagnostics, environment monitoring, and other types of research.
Experimenting With the Connections
When we unplugged the display from the circuit board, the screen on the machine went blank. This is because the screen was no longer connected to the power in the circuit board.
When we unplugged the white wire that connects circuit board to heating board, the machine the top tempurature reading went from 25 degrees Celcius to -40 degrees Celcius. The tempurature sensor was unable to read the tempurature.
Test Run
The date of the PCR test run was 3/20/13. It went through 15 of the 35 cycles. We ensured that in each cycle that it was at the correct temperature as dictated by the protocol. The PCR machine was successful in going through the different cycles at the right time frames and correct temperatures. The PCR test run was a pass and the PCR machine was marked as such.
Protocols
Thermal Cycler Program
Protocol:
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
Anneal at 57 degrees Celsius for 30 seconds
Extend at 72 degrees Celsius for 30 seconds
Final Step: 72 degrees Celsius for 2 minutes
Final Hold: 4 degrees Celsius
DNA Sample Set-up
Positive Control: disease DNA Tube Label: A1
Patient 1, Replicate 1 Tube Label B1
Patient 1, Replicate 2 Tube Label B2
Patient 1, Replicate 3 Tube Label B3
Negative Control: non-disease DNA Tube Label A2
Patient 2, Replicate 1 Tube Label C1
Patient 2, Replicate 2 Tube Label C2
Patient 2, Replicate 3 Tube Label C3
DNA Sample Set-up Procedure
Step 1: Assign the patients ids and create tube labels. Also create tubes labels for one positive control and one for a negative control.
Step 2: Add 50 ul of the PCR reaction mix to each tube.
Step 3: Add 50 ul + DNA/primer mix to the tube labeled as the positive control.
Step 4: Add 50 ul - DNA/primer mix to the tube labeled as the negative control.
Step 5: Add 50 ul of the patient 1 replicates to a tube labeled for patient 1.
Step 6: Repeat step 5 for the other two patient 1 replicates.
Step 7: Add 50 ul of the patient 2 replicates to a tube labeled for patient 2.
Step 8: Repeat step 7 for the other two patient 2 replicates.
Step 9: Close the lids for each PCR reaction tube.
Step 10: Place the tubes into the heating block of the PCR machine.
Step 11: Set PCR machine to correct temperatures and number of cycles.
PCR Reaction Mix
What is in the PCR reaction mix? The PCR reaction mix is made up of 50 ul mix with Taq DNA polymerase, MgCl2, and dNTP's. This mix includes the proteins and nucleotides necessary to complete PCR. This mix can be used for each reaction.
DNA/ primer mix
What is in the DNA/ primer mix? The DNA/primer mix is made up of 50 ul mix with the same forward primer and reverse primer and each has a different template DNA. This is made new for each PCR because it is specific to each reaction.
Research and Development
PCR - The Underlying Technology Reagents of PCR
Template DNA: The Taq polymerase uses this strand of DNA to create an identical strand of DNA. It is copied during the synthesis of mRNA.
Primers: Primers are short pieces of lab made DNA that have a sequence of nucleotides. One of the primers attaches to the top of the DNA strand and other is a reverse primer and it connects at the bottom of the other DNA strand.
Taq Polymerase: These are proteins that copy the DNA after attaching to the primer.
Deoxyribonucleotides (dNTP's): This is a mix of DATP, DTTP, DCTP, and DGTP, which are nucleotides that the polymerase uses to build the new strand of DNA.
Steps of Thermocycling
Heated Lid: 100 degrees Celsius
Initial Step: 95 degrees Celsius for 2 minutes (double helix gets separated, creating 2 single stranded DNA molecules)
Number of Cycles: 35
Denature at 95 degrees Celsius for 30 seconds (double helix gets separated, creating 2 single stranded DNA molecules)
Anneal at 57 degrees Celsius for 30 seconds (the primers bond to the strands bracketing the sequence to be copied)
Extend at 72 degrees Celsius for 30 seconds (Taq polymerase binds to the sequences to build the new strands)
Final Step: 72 degrees Celsius for 2 minutes
Final Hold: 4 degrees Celsius (Keeps it cool so it doesn't continue to replicate)
SNP-Single Nucleotide Polymorphism Background
Int his section, the team looked at how the PCR machine is used to detect a disease-associated DNA sequence (SNP). A nucleotide is a subunit of nucleic acids such as DNA and RNA. It is made up of a nitrogenous base, a five carbon sugar and a phosphate group. The nucleotides are the building blocks of the DNA and RNA. A polymorphism is the presence of a genetic variation within a population. A single nucleotide polymorphism is when a single nucleotide differs from others in that particular population. A PCR can be used to detect these SNPs.
Example SNP
An example of an SNP is the rs237025. This variation is found in homo sapiens. The variation is found on the chromosome 6:149721690. The genes associated with this SNP are the Sum04 and Tab2. The diseases linked to this SNP is type 1 diabetes. The team looked further into the Sum04 gene. It stands for Small ubiquitin-like modifier 4. It is the linked to the molecular function of missense and config reference. The non disease allele is GTG while the disease allele is CTG. An allele is one or more alternate forms of a gene that arise by mutation. The numerical position of the SNP is 149721690.
Design of a Disease Sequence-Specific Primer Pair
In order to use the PCR machine, a disease-allele-specific forward primer and a reverse primer must be created. The disease-allele-specific forward primer is (starting at the 5' end):AACCACGGGGATTGTCAGTG. The reverse primer, necessary for amplifying DNA, (starting at the 5' end) is: TTGGTGCCCCTAACAGTCAC. If the either primer does not bind 100% to the template then the PCR result will be blank. It will not be any different from when it started
BME 100 Spring 2014
