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.
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G3 +
Positive control
none
G3 -
Negative control
none
G3 1-1
Patient 1, replicate 1
69628
G3 1-2
Patient 1, replicate 2
69628
G3 1-3
Patient 1, replicate 3
69628
G3 2-1
Patient 2, replicate 1
57443
G3 2-2
Patient 2, replicate 2
57443
G3 2-3
Patient 2, replicate 3
57443
DNA Sample Set-up Procedure
Move the DNA sample into a PCR tube in ice using a micropipette (use a new pipette tip for every transaction)
Move Primer 1 into the PCR tube with the micropipette and then Primer 2
Add a solution of Nucleotides into the PCR tube using the micropipette
Add a solution of DNA Polymerase into the PCR tube
Place the tubes in the thermal cycler
OpenPCR program
Heated Lid: 100℃
Initial Step: 95℃ for 2 min
Number of Cycles: 25
Cycle: Denature at 95℃ for 30 seconds, Anneal at 57℃ for 30 seconds, and extend at 72℃ for 30 seconds
Final step: 72℃ for 2 min
Final hold: 4℃ for 2 min
Components
In replicating DNA, there first needs to be a DNA sample that will be copied. In PCR this is called template DNA. It is typically not an entire DNA strand, as they are very long, but a segment of DNA. Additionally, there need t =o be building blocks to make new DNA. These are called deoxyribonucleotides (dNTP's) and they are the base pairs that make up all DNA. Without these, there would be nothing to actually create the DNA. However, these building blocks will not spontaneously bind the the DNA placed in a solution with them. In order for the dNTP's to bind, DNA polymerase must be in place. Taq polymease is most commonly used in PCR because it is extremely resistant to heat. However there is one more problem. In order for DNA to be replicated it must be single-stranded, however DNA polymerase will only bind to a double-stranded DNA. To combat this, primers must be used. They are short segments of DNA that are often custom made for each DNA strand that bind to the 3' end of DNA strands. They provide for a binding site for the DNA polymerase and allow for DNA replication.
The Process
The first step of PCR is called initialization. The solution is held at 95 degrees Celsius for 2 minutes. During this step, the DNA polymerase is activated. This step is not always necessary, but most DNA polymerases need to be heat activated. Next, the solution is held at 95 degrees Celsius for 30 seconds. This serves to denature the DNA and split it from two strands to one. The DNA is then read for the primers to attach to the DNA. This is done by cooling the solution to 57 degrees Celsius for 30 seconds in a step called annealing. Once the primers are attached, the solution is heated to 72 degrees Celsius for 30 seconds. In this step, the DNA polymerase attaches to the primer and starts to synthesize DNA. It is called extension and the dNTP's are placed in the correct positions in the 5'to 3' direction.
After however many rounds of these steps are done, a final step of keeping the solution at 72 degrees Celsius for 2 minutes is performed. This allows for any leftover single-stranded DNA to complete the extension process. Once this is complete, the solution is held at 4 degrees Celsius for storage.
Base Pairing
The dNTP's in PCR will not randomly pair with the single-stranded DNA. When the DNA polymerase is building the new strand, the base Adenine will always trigger the base Thymine and the base Guanine will always trigger the base Cytosine and vice verse. A and T always go together and C and G always go together
SNP Information & Primer Design
Background: About the Disease SNP
Single nucleotide polymorphisms are the case where a genetic variation has presented itself on a nucleotide: a compound which consists of a nucleoside linked to a phosphate group; Nucleotides form the basic structural unit of nucleic acids such as DNA.The disease SNP, ENPP1, variation is found in Homo sapiens and is located on chromosome 6:131851228.The disease-associated allele contains a change in codon positioned at 131,851,288 and changes from AAG to CAG.The current clinical significance of this SNP includes a Benign risk factor with another allele. Conditions most associated to the SNP include bone disorders in type 2 diabetes and sickle cell anemia. ENPP1 stands for Ectonucleotide pyrophosphatase/phosphodiesterase 1 and its functions include the hydrolyzation of nucleoside 5’-triphosphates to their corresponding monophosphates, ATP binding, and NADH-pyrophosphatase activity. When ENPP1 is affected, it cannot continue its normal functions the organism can undergo drastic side-effects.
Primer Design and Testing
In order for PCR to amplify DNA, it will need two primers. One of the primers is the non-disease forward primer, which is 5'-TTCAGATGACTGCAAGGACA. The other non-disease reverse primer for the PCR is 5'TGTTTAAAAGTTTCTTTAAT. The genetic variation SNP is accounted for two primers, one of them being the disease forward primer: 5'-TTCAGATGACTGCAAGGACC, and the other being the disease reverse primer: 5'-TGTTTAAAAGTTTCTTTAAT. The numerical position exactly 200 bases to the right of the disease SNP is 131,851,428. The primer for the Nondisease sequence was calculated and resulted in the UCSC In-Silico PCR with confirmation that the primers would work. The Disease-specific primer resulted in "no matches" due to the single nucleotide polymorphism that arises in the sequence, and causes it to differentiate from the Non-Diseased sequence.
BME 100 Spring 2018
