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
G14 +
Positive control
G14 -
Negative control
G14 1-1
Patient 1, replicate 1
79929
G14 1-2
Patient 1, replicate 2
79929
G14 1-3
Patient 1, replicate 3
79929
G14 2-1
Patient 2, replicate 1
84911
G14 2-2
Patient 2, replicate 2
84911
G14 2-3
Patient 2, replicate 3
84911
DNA Sample Set-up Procedure
Collect all the required materials needed for the experiment. These are listed in the “Materials” section.
Cut the linked PCR tubes in half, it will result in two strips of PCR tubes and four linked tubes per each strip.
Label the side of the tubes with the group's specific labels with a marker.
Put the tubes into a rack.
Put 50 μL of PCR reaction mix in the positive control PCR tube and discard the pipette tip.
Use a new tip and put the corresponding DNA/primer mix into the positive control tube.
Repeat the previous 2 steps for all of the remaining test tubes.
Secure the tubes' lids.
Place the PCR tubes in the heating block of the PCR machine.
Result will be displayed after the cycle is complete.
Clean up lab station, following guidelines for proper biomaterial disposal.
OpenPCR program
HEATED LID: 100°C
INITIAL STEP: 95°C for 2 minutes
NUMBER OF CYCLES: 25
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and
Extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
What is the function of each component of a PCR reaction?
The Polymerase Chain reaction has several components that each have a unique function. Template DNA is the region of DNA that will be amplified by the PCR process. In order to begin the multiplication of the Template DNA, two different primers need to be added to the reaction. Both primers are short sequences of nucleotide with opposite configurations that begin the creation of new DNA from opposite ends of the Template DNA strand. The actual replication of the DNA is carried out by a polymerase enzyme found in thermophilic bacteria, Thermus aquaticus, namely called Taq Polymerase. Taq Polymerase adds free deoxyribonucleotides, which are individual base units of DNA, to the Template DNA strands to create two copies of the original DNA.
What happens during thermal cycling?
During thermal cycling, the process that allows the polymerase chain reaction to occur, the DNA and the other components of the reaction are cycled through different temperatures for specific amounts of time. The initial step of thermal cycling heats the reaction chamber to 95 degrees centigrade for 2 minutes to activate the Taq Polymerase. For the Taq Polymerase to function, the Template DNA is denatured and broken into the two single-stranded DNA molecules. This process happens at 95 degrees centigrade for 30 seconds. Next, the process of annealing occurs at 57 degrees for 30 seconds. Annealing is the step of thermal cycling in which short DNA primers bind to the targeted sequence of DNA (in this case the SNP) and delineate the area of DNA that is to be copied. Typically, DNA would try to bind together, but the primers prevent this from happening. Soon after annealing has occurred, extension occurs for 30 seconds at 72 degrees. At this point in thermal cycling, Taq Polymerase starts adding complimentary base pairs to the single-stranded DNA using the primers as a starting point. The processes up to this point are repeated for many cycles, and the desired fragments of DNA appear as two paired strands containing the target sequence. The final step consists of the temperature standing at 72 degrees for 2 minutes which ensures that the nucleotides are bound. Finally, the reaction chamber is held at 4 degrees so that the product of the reaction can be stored for a while.
Which bases are paired during base pairing?
In the process of base-pairing, each deoxyribonucleotide pairs with only one other deoxyribonucleotide by hydrogen bonding. Adenine (A) always pairs with Thymine (T), and Cytosine (C) always pairs with Guanine (G).
During which two steps of thermal cycling does base-pairing occur?
Base pairing occurs in two steps of thermal cycling: annealing and extension. During the annealing phase short DNA primers attach to the targeted DNA and serve as starting points for the replication. When the temperature is raised in the extension step, Taq Polymerase actively matches the base pairs to form nucleotides, extending the primers along the single strand DNA.
SNP Information & Primer Design
Background: About the Disease SNP
A single nucleotide polymorphism is a variation of a single nucleotide (the basic structural units of DNA) on a specific location in the genome. The variation rs721710 is found on chromosome 12 in Homo sapiens. This SNP clinical significance is listed as an allele with uncertain significance. rs721710 is associated with conditions such as Parkinson's Disease. Essentially, the significance of this variation is uncertain, but it has a correlation with known disease. LRRK2, which stands for leucine-rich repeat kinase 2, is chiefly involved in ATP binding, GTP binding, and GTP-dependent protein kinase activity. When LRRK2 is subject to mutation, these functions begin to fail, thus causing genetic diseases such as Parkinson's Disease. The codon related to the diseased allele of LRRK2 is GAG.
Primer Design and Testing
When inputting the non-disease forward primer 5’-TTAAGTGACTTGTACTTTGT-3’ and the non-disease reverse primer 5’-TGAAGCTCTTCAAGTAGTCT-3’ into the UCSC In-Silico PCR webpage, the correct 220bp sequence was obtained.
When inputting the disease forward primer 5’-TTAAGTGACTTGTACTTTGA-3’ and the disease reverse primer 5’-TGAAGCTCTTCAAGTAGTCT-3’ into the UCSC In-Silico PCR webpage, no results were returned.
BME 100 Fall 2018
