PCR reaction mix, 8 tubes, 50 µL each: Mix contains Taq DNA polymerase, MgCl, and dNTP's
DNA/ primer mix, 8 tubes, 50 µL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer.
A strip of empty PCR tubes
Disposable pipette tips: only use each only once. Never reuse disposable pipette tips.
Cup for discarded tips.
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G5 +
Positive control
none
G5 -
Negative control
none
G5 1-1
Patient 1, replicate 1
29596
G5 1-2
Patient 1, replicate 2
29596
G5 1-3
Patient 1, replicate 3
29596
G5 2-1
Patient 2, replicate 1
61164
G5 2-2
Patient 2, replicate 2
61164
G5 2-3
Patient 2, replicate 3
61164
DNA Sample Set-up Procedure
Obtain a DNA sample and transfer the extracted DNA into a PCR tube.
Add Primer 1 into the PCR tube with extracted DNA.
Add Primer 2 into the PCR tube with extracted DNA and Primer 1.
Add nucleotides to the PCR tube with DNA and Primers 1 and 2
Lastly, add DNA Polymerase to the PCR tube
Once filled with all of the contents, place the PCR tube, containing DNA, Primers 1 and 2, nucleotides, and DNA Polymerase, into the thermocycler.
OpenPCR program
HEATED LID: 100°C
1. INITIAL STEP: 95°C for 2 minutes.
2. NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds.
Anneal at 57°C for 30 seconds.
Extend at 72°C for 30 seconds.
3. FINAL STEP: 72°C for 2 minutes
4. FINAL HOLD: 4°C
Research and Development
The Underlying Technology
PCR - Important Components
Polymerase Chain Reaction (PCR for short) is an easy and inexpensive way to focus in on a segment of DNA and be able to copy it up to a billion times over. The segment of DNA that you wish to have copied is known as the template DNA. Primers are then attached to the template DNA that is to be copied in very specific spots. There are two primers necessary to complete the PCR, one primer that attaches to the top strand of DNA, and the other will attach to the bottom strand. Once attached, these primers will show the DNA polymerase where to start copying the DNA, and in the case of PCR, a special polymerase called Taq polymerase will be used. When the Taq polymerase begins to read the strand of DNA, it brings in deoxyribonucleotides (dNTP's) to create the complimentary strand of DNA, which are composed of 4 dNTP's: Adenine, Thymine, Guanine, and Cytosene. Thermal Cycling
Thermal Cycling is the process that is used to copy the template DNA over and over again. It starts with the template DNA and all other necessary materials heated up to 95°C for 2 minutes. Once the DNA gets hot enough, the double helix will begin to unwind, and the hydrogen bonds that hold the strands together begin to break due to the high amount of heat, and the strands will separate. This process is known as denaturing and it takes 30 seconds to complete this process. After this occurs, the solution is then cooled down to a temperature of 57°C for 30 seconds, and in this time, a process called annealing takes place, which allows for the two primers to be able to attach to the specific locations on the strand of DNA that is to be copied. The heat is then raised to 72°C for 30 seconds to allow for Extension to occur, which is when the Taq polymerase attaches to the primers and reads the DNA strand to create complimentary strands using dNTP's that are in the solution. After the extension process, the solution is then held at 72°C for 2 minutes to allow for extra time to create the new strands. Once the PCR is finised, the DNA will be held at a temperature of 4°C. https://www.clinisciences.com/en/buy/cat-conventional-pcr-3473.html
SNP Information & Primer Design
Background: About the Disease SNP
SNP (Single nucleotide polymorphism) is a variation of the 12th chromosome in Homo sapiens (humans). Clinically speaking, the significance of this variation is uncertain, however Parkinson’s Disease is often linked to the variation. This variation affects the gene in the 40315266 position and the disease associated allele contains GAG, rather than GTG. An allele is a different or alternative form of a gene. The LRRK2 (leuchine rich repeat kinase 2) helps to encode a protein that is largely present in the cytoplasm. This protein aides in ATP Binding, GTP Binding, and GTP dependent protein kinase activity.
Primer Design and Testing
The non-disease forward primer the group designed was 5’-ttaagtgacttgtactttgt. The group obtained these results by writing down the sequence of the 20 bases from the position of SNP. The non-disease reverse primer the group designed was 5’ -tgaagctcttcaagtagtct. This was found by going along the bottom row of the DNA strand and to the right 200 bases from the SNP location. Once the group reached that position, the group counted backwards 20 bases and wrote down those 20 bases. For the disease forward primer, all that was needed was to change the final base to it's corresponding base. This gave the disease forward primer of 5’-ttaagtgacttgtactttga. To obtain the disease reverse primer, the non-disease reverse primer is just copied, giving 5’ -tgaagctcttcaagtagtct. After running the non-disease primers through the website, the result the group was given was the 220 bp sequence from the chromosome found earlier in the lab, confirming that the non-disease primer would in fact work. After running the disease primers through the website, the result obtained was no match, confirming that the disease primers were correct.
Results from non-disease primer.Results from disease primer.
BME 100 Fall 2018
