LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2 , 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
- Cup for discarded tips
- OpenPCR machine: shared by two groups
- DNA samples from 2 patients identified below
PCR Reaction Sample List
||PCR Reaction Sample
||Patient 1, replicate 1
||Patient 1, replicate 2
||Patient 1, replicate 3
||Patient 2, replicate 1
||Patient 2, replicate 2
||Patient 2, replicate 3
DNA Sample Set-up Procedure
- Collect all the required materials for Lab A
- Take the strip of empty PCR tubes and cut them so you have two strips of four linked tubes. This is for the machine.
- Label with a black marker the sides of the empty tubes with the Tube labels your group has created. Do not label the lids of the tubes.
- Place the tubes in a rack.
- Start with the empty tube labeled positive control. Use the pipette to transfer 50 μL of PCR reaction mix into this empty tube. Get rid of the disposable tip into the collection cup. Do not re-use tips.
- Using a new pipette tip, transfer the positive control DNA/primer mix into the same tube. Your total volume in you positive control PCR reaction is now 100 μL.
- Repeat steps 5 and 6 for the negative control, patient 1 replicates 1,2 and 3, and patient 2 replicates 1,2, and 3. Using the appropriate DNA/ primer mix for the corresponding tubes. When finished, all of your labeled tubes should have DNA/ primer mix plus PCR mix, resulting in a 100 μL complete PCR reaction in each tube.
- Close all of the lids tightly on your PCR reaction tubes.
- Take your tubes to your assigned PCR machine. Place the tubes into the slots in the heating block. Do not start until all 16 slots are filled(multiple groups are needed). If you are unsure ask a TA for help.
- Step a: Make sure you have installed Adobe Air and the OpenPCR onto your computer
- Step b: Connect your OpenPCR machine to a wall outlet and plug it into your computer via a USB before step 3.
- Step c: Turn on your OpenPCR device and start the OpenPCR app.
- Step d: Set up the Heated Lid
- Unscrew the black knob counter-clockwise all the way. Open the lid and put several empty PCR tubes into OpenPCR's tube block
- Close the lid and screw the knob clockwise to lower the heated lid. Watch carefully, when the front of the lid starts to lift up it is at the proper height, stop.
- Add a drop of water to an empty PCR tube and run a PCR experiment for a few cycles. After the cycles are completed, open the lid and check that there is not much condensation on the top of the tube.
- The heated lid set up is complete.
- Step e: On the home screen click "Add new experiment" and on the blank protocol screen click the "More Options" button at the bottom.
- Step f: Fill in the following temperatures, time, and number of cycles as follows:
- Heated Lid: 100 C
- Initial Step: 95 C for 2 minutes
- Number of Cycles: 25
- Denaturing temp: 95 C
- Denaturing time: 30 secs
- Final Step: 72 C for 2 minutes
- Final Hold: 4 C
Research and Development
PCR - The Underlying Technology
Components to reactions
The first component is a Template DNA, which is literally a template for the DNA polymerase to attach to and start adding base pairs upon.
The second component of this reaction is Primers. Primers are short pieces of DNA, usually custom-built in a laboratory to have any sequence of nucleotide. They serve as the starting place on the DNA template for the DNA polymerase to attach itself. The third component is a Taq polymerase. Although it also attaches to the DNA template for copying DNA, tag polymerase is special from normal polymerase as it does not break down and functions in much higher temperatures than normal. The last and fourth component are the Deoxyribonucleotides. They are single units of DNA made up of nitrogenous base, a phosphate group, and a deoxyribose sugar.
What Happens to the Components during the Thermocycling
During the initial step of 95 C for 2 minutes, the PCR tubes filled with DNA, primers, nucleotides, and the polymerase are heated. This causes the DNA to denature or to split in half along the double helix. The next 25 cycles between 95 and 57 C are to allow for the primers to attach to the split DNA templates and for the Tag polymerase to start copying the new DNA sequences without any competition from normal polymerase. They use the deoxyribonucleotides to build the new and improved DNA. The repeating cycle is to encourage as much splitting and subsequent rebuilding as possible of the new DNA. During the Final step of 72 C for 2 minutes, base pairing occurs. This means that the new coded DNA strands are rebound into functioning double helixes. The correct binding pairs for the DNA nucleotides are as follows: Adenine - thymine bind together and Cytosine - Guanine bind together. The last step of the thermocycling is the final hold at 4 C which is stop the process and preserve the compiled DNA.
SNP Information & Primer Design
Background: About the Disease SNP
Single Nucleotide polymorphism, abbreviated as SNP, is a collective term for simple genetic variations whether these variations are nucleotide substitutions, deletions, insertions, or repeats. Nucleotides are defined as a structural building block in the composition of DNA and RNA. It consists of one phosphoric acid, a molecule of sugar, and a base: adenine, thymine, guanine, and cytosine. This variation, SNP is very frequent as it occurs in every 1 out of 1,000 bases.
The variation rs721710 is found in homo sapiens. The chromosome variance is located on 12:40315266. he clinical significance of this SNP is uncertain; however, on PubMed, it is stated that there is a link between this variation and Parkinson's Disease. The DNA sequence of the SNP is known to LRRK2, the leucine-rich repeat kinase 2 gene. This gene is known to be linked to familial Parkinsonism. There was a study conducted which allowed for LRRK2 to be genotyped in patients with Parkinson's. The function of LRRK2 gene is active in the brain and responsible for making a protein, dardarin, which is responsible for interactions with other proteins, transmitting signals in the brain, or assembling a cell's structure.
An allele is an alternative form of a gene that arises from mutation. The non-diseased allele is GTG and the disease associated allele contains GAG. The numerical position is 40315266.
Primer Design and Testing
The non-diseased forward primer is 5'T-T-A-A-G-T-G-A-C-T-T-G-T-A-C-T-T-T-G-T 3' while the diseased forward primer is 5' T-T-A-A-G-T-G-A-C-T-T-G-T-A-C-T-T-T-G-A 3'. The numerical position is 40315466 after exactly 200 bases have been added. After this has been added, the non-diseased reverse primer is 5' T-G-A-A-G-C-T-C-T-T-C-A-A-G-T-A-G-T-C-T 3' while the diseased reverse primer is 5' T-G-A-A-G-C-T-C-T-T-C-A-A-G-T-A-G-T-C-T 3'.