NOTE: only use each only once. Never reuse disposable pipettes. If you do, the samples will become cross‐contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G17 +
Positive control
none
G17 -
Negative control
none
G17 1-1
Patient 1, replicate 1
38060
G17 1-2
Patient 1, replicate 2
38060
G17 1-3
Patient 1, replicate 3
38060
G17 2-1
Patient 2, replicate 1
54209
G17 2-2
Patient 2, replicate 2
54209
G17 2-3
Patient 2, replicate 3
54209
The positive control is DNA positive for the disease SNP (has it)
The negative control is DNA negative for the disease SNP (does not have it)
DNA Sample Set-up Procedure
Obtain the materials listed above.
Cut the empty PCR tube strip in half, obtaining two links of four tubes.
Label the sides of the empty tubes with a black marker, making sure they match the labels in the table above.
Place tubes in PCR tube rack.
For the tube labeled as POSITIVE CONTROL: pipette 50 microliters of the PCR reaction mix into tube, discard tip. Pipette 50 microliters of the positive control DNA/primer mix into tube, discard tip. Total volume should now be 100 microliters.
Repeat the above step for the NEGATIVE CONTROL, PATIENT 1 REPLICATES 1, 2, 3, and PATIENT 2 REPLICATES 1, 2, 3. Each sample will have a corresponding DNA/Primer mix - use this. Be sure to discard pipette tips each time to prevent cross-contamination. Every tube should contain 100 microliters of fluid.
Close lids tightly and take to assigned PCR machine.
Place tubes into the machine in the heating block. 16 slots must be filled before running the machine - share with other groups.
CLEAN-UP: return reusable material as instructed and dispose of biological waste in a biohazard bag. Clean area.
Note any deviations (especially in labeling) in lab report.
OpenPCR program
Thermal Cycler heating and cooling values:
Heated Lid: 100 C
Initial Step: 95 C for 2 minutes
Number of Cycles: 25
Denature: 95 C for 30 seconds
Anneal: 57 C for 30 seconds
Extend: 72 C for 30 seconds
Final Step: 72 C for 2 minutes
Final Hold: 4 C (infinite)
Reference: BME 100 lab Workbook: DNA labs, 2018. Supplied by Dr. Haynes and Dr. Garcia. Arizona State University - School of Biological and Health Systems Engineering.
Research and Development
PCR - The Underlying Technology
FUNCTIONS OF PCR COMPONENTS
The Template DNA is a double-stranded (DS) DNA molecule that is used as a foundation to make copies of a specific sequence it contains. It is separated into two strands, to which the primers attach to at 5-prime end in order to initiate the reaction. Primers are sequence-specific DNA strands that bind to the separated template DNA strands and call for the Taq Polymerase to bind and replicate the template DNA. The polymerase cannot perform without the primers, as it can only attach nucleotides to a preexisting DNA strand.
Taq polymerase is a polymerase isolated from a thermophile bacteria, and so involves a higher temperature to activate. This ensures that the reaction does not start prematurely. A polymerase attaches to a single DNA strand using the primer, and begins to take "floating" deoxyribonucleotides within the solution and attaches them to the DNA strand. This results in a new double-stranded DNA molecule, just like the template DNA strand. The cycle then repeats itself within a ThermalCylcer, resulting in exponential growth of the amount of the original template DNA amount.
Deoxyribonucleotides (dNTPs) are molecules that make up DNA: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). Each of these molecules form a sugar-phosphate backbone of the DNA strand with their neighbor, and reach across to bond with each other to form a double-stranded DNA helicase. The bonds between each deoxyribonucleotides are very specific; adenine always binds with thymine, and cytosine always binds with guanine. These molecules are what create a DNA strand's sequence.
THE COMPONENTS IN THERMAL CYCLING
Thermal cycling is the process of heating and cooling the PCR mixture in order to exponentially increase the amount of a template DNA strand. There are six traditional steps: initializing, denaturing, annealing, extending, final, and final hold.
The initial step heats and hold the mixture at 95 C for 2 minutes. This is the initial denaturing step, where the double-stranded DNA separates into single-stranded DNA. It is held for two minutes to make sure that each template DNA strand separates and is available for the primers to reach. It may also serve as an activation step for hot-start polymerases. The denaturing step, including what occurs in initializing, separates the two strands of DNA by disrupting the hydrogen bonding between the adenine - thymine and cytosine - guanine bonds. The results are single strands of the template DNA.
The annealing step lowers the mixture temperature to 57 C, and allows the primers to attach to the DNA strands. The lower temperature allows for the hydrogen bonds between the base pairs of the DNA strand and primer to anneal, or come together.
The extending step raises the temperature slightly to 72 C, which allows for the Taq polymerase to "extend" the hybridized primers. This is the process of taking an appropriate floating dNTP, matching it with the nucleotide the polymerase is on, and creating the bond between them. This continues to follow the adenine - thymine and cytosine - guanine bonding rule. The final step is a continuation of the last extending step (the denature, annealing, and extending steps are often repeated many times over) and allows for the DNA strands to "settle" into double-stranded DNA molecules that are used for manipulation after the PCR sequencing is complete.
The final hold decreases the temperature to 4 C and prevents any more reactions from occuring. It holds the new PCR products until someone can use them them.
BASE PAIRS AND THERMAL CYCLING
This image shows the base pairing using hydrogen bonds between Adenine - Thymine and Cytosine - Guanine. Base-pairing occurs during the annealing and extending steps of PCR.
Reference for image:
Van der Wijst, T., Guerra, C. F., Swart, M., & Bickelhaupt, F. M. (2006). Performance of various density functionals for the hydrogen bonds in DNA base pairs. ScienceDirect, 426(4-6), 415-421. doi:10.3897/bdj.4.e7720.figure2f
SNP stands for "Single Nucleotide Polymorphism," and is a change in one nucleotide of a DNA sequence. The SNP rs721710 is a variation found in humans (Homo sapiens) located on chromosome 12:40315266. The clinical significance of this SNP is uncertain, and familial parkinsonism is linked to it. It is the gene LRRK2, or leucine-rich repeat kinase 2. This gene is involved in ATP binding, GTP binding, and kinase activity. An allele is a version of a gene sequence on a chromosome. The disease - causing allele for this SNP contains the codon "GAG." The non-disease-causing codon is "GTG." The numerical position of this SNP is at 40315266.
Primer Design and Testing
Based on the information above, we isolated forward and reverse primer sequences for the disease-causing and non-disease-causing alleles. We then tested this information in UCSC's In-Silico PCR website. Our non-disease-causing primer worked, giving us the sequence of the desired gene. The disease-causing primer did not work, as the primers for the diseased allele would not anneal to a sequence within the database - the normal gene.
BME 100 Fall 2018
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