PCR reaction mix, 8 tubes, 50 µL each: Mix contains Taq DNA polymerase, MgCl2, 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. 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
G10 +
Positive control
none
G10 -
Negative control
none
G10 1-1
Patient 1, replicate 1
38679
G10 1-2
Patient 1, replicate 2
38679
G10 1-3
Patient 1, replicate 3
38679
G10 2-1
Patient 2, replicate 1
72739
G10 2-2
Patient 2, replicate 2
72739
G10 2-3
Patient 2, replicate 3
72739
DNA Sample Set-up Procedure
Check that your lab group has all required materials.
Slice the strip of empty PCR tubes in half to obtain two strips of four linked tubes, so that all tubes fit into the machine.
Label the sides of the empty tubes with a black marker with the labels from the above table.
Put the tubes in a rack.
Using the empty tube previously labeled as the positive control, transfer 50 µL of PCR reaction mix into the tube. Make sure to be using proper pipetting technique! Place the disposable tip into the collection cup afterwards. Be cautious not to re-use tips and cross-contaminate the samples!
Using a new pipette tip, put both the positive control DNA and the primer mix into the same tube. The total volume in the positive control PCR reaction tube should now be 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. Once all of the negative controls are completed, mix the appropriate DNA and primer mix for each of the tubes. When all of the tubes are correctly mixed, they should all be labeled and contain DNA, primer mix, and PCR mix, with a total volume of 100 µL of complete PCR reaction.
Tightly secure the lids on the PCR reaction tubes.
Bring all of the tubes over to your assigned PCR machine, and place the tubes into the slots in the heating block. Do not start the machine until all 16 slots are filled, as multiple groups will need to run reactions at the same time. If you're not sure if it is okay to start a run, ask a TA for help.
OpenPCR program
HEATED LID will be kept at 100°C
FIRST STEP: heat at 95°C for 2 minutes
NUMBER OF CYCLES: 25
- For each cycle, follow the following model
- Denature at 95°C for 30 seconds
- Anneal at 57°C for 30 seconds
- Extend at 72°C for 30 seconds
LAST STEP: Put temperature at 72°C for 2 minutes
FINAL HOLD: 4°C
Research and Development
PCR - The Underlying Technology
1)
Template DNA- At the start of the reaction, high temperature is applied to the DNA molecule causing it to separate the strands from each other.
Primers- Through the process of base pairing, a primer will attach to the top of a strand and another primer will attach to the bottom of the strand, to assist DNA Polymerase to copy the strand of DNA.
Taq Polymerase- Enzyme that is used to replicate DNA.
Deoxyribonucleotides (dNTP's)- The building blocks of DNA, used to create the strands of DNA.
Genetic Science Learning Center. (2013, February 14) PCR. Retrieved October 18, 2016.
Khalsa, Guruatma. "ASU School of Life Sciences | Ask A Biologist." Mama Ji's Molecular Kitchen. N.p., n.d. Web. 18 Oct. 2016.
2)
INITIAL STEP: 95°C for 3 minutes:- DNA begins to separate and unwind.
Denature at 95°C for 30 seconds:- Hydrogen Bonds holding the double helix together are broken down and makes single-stranded DNA molecules.
Anneal at 57°C for 30 seconds:- Two primers will attach to either the top or the bottom of each strand of DNA.
Extend at 72°C for 30 seconds:- Taq Polymerase bind to the primers and begin to create nucleotides for replication.
FINAL STEP: 72°C for 3 minutes:- During this step, the single-stranded DNA are fully extended. Taq polymerase is added until the end of the strands.
FINAL HOLD: 4°C:- This is where the reactions comes to an end and shuts off the taq polymerase.
4)
Base Pairing occurs during the annealing and extension steps of thermal cycling. In the process of annealing, the system is cooled so that the target DNA can bind with the short DNA primers to start replication. During the extension process, the system is heated up 15°C, and taq polymerase match the base primers with their other pairs and extend the primers, forming a new nucleotide strand of the desired target sequence.
The assigned SNP is exclusively found in homo sapiens (humans), in the chromosome 4:113367751. It is a pathogenic mutation linked to cardiac arrhythmia syndrome caused by the loss of ankyrin-B function (abbreviated ANK2). The gene ontology included with this SNP includes, among others, ATPase binding, cytoskeletal adaptor activity, and enzyme binding. The disease-associated-allele (an allele is a variation in a gene that can cause phenotypic variation, such as the alleles for hair color or eye color) has been found to contain the codon ATC, deviating from the standard CTC. The particular SNP is found at the numerical position of 112,706,083..113,383,740.
Primer Design and Testing
The non-disease forward primer is derived from the numerical position of the SNP. As for the non-disease reverse primer, it is derived from 200 bases to the right of the SNP position. The non-disease forward and reverse primers were checked to see if they are acceptable primers through a online source. The online source proved that the non-disease forward and reverse primers are acceptable because they received the desired outcome of a 220 bp sequence.
BME 100 Fall 2016
