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
G9 +
Positive control
none
G9 -
Negative control
none
G9 1-1
Patient 1, replicate 1
20925
G9 1-2
Patient 1, replicate 2
20925
G9 1-3
Patient 1, replicate 3
20925
G9 2-1
Patient 2, replicate 1
23743
G9 2-2
Patient 2, replicate 2
23743
G9 2-3
Patient 2, replicate 3
23743
DNA Sample Set-up Procedure
Move the DNA extracted from the patient to a PCR reaction tube.
Add Primer 1 to the PCR reaction tube with the DNA.
Add Primer 2 to the PCR reaction tube with the DNA and Primer 1.
Add nucleotides to the PCR reaction tube with the DNA, Primer 1, and Primer 2 so that there are materials to build complementary DNA fragments during the PCR process.
Add DNA polymerase to the PCR reaction tube to create the DNA fragments.
Place the PCR reaction tube into the thermal cycler.
OpenPCR program
Set the thermal cycler lid to 100° C. Then, set the cycler to 95° C for 2 minutes. After this, set the cycler to 25 cycles of denaturation at 95° C for 30 seconds, annealing 57° C for 30 seconds, and extending at 72° C for 30 seconds. Lastly, hold the cycler 72° C for 2 minutes, and then at 4° C.
Research and Development
PCR - The Underlying Technology
Basic Materials/Components
There will be a couple of components used during the PCR Reaction. Each one of these components has a specific purpose in the reaction process. The first component is template DNA is the original DNA which is used to produce the desired complementary DNA fragments that will be amplified in the PCR reaction. The next components are primers, which are the short segments of DNA added that provide a starting point for where replication is supposed to occur by binding to the template DNA at the specific complementary matches, marking where the Taq Polymerase are supposed to work. Then, Taq Polymerase is necessary as the enzyme that takes nucleotides and links them together in the correct sequence to create complementary DNA fragments to the template DNA. Finally, dNTPs (deoxyribonucleotides) are the nucleotides and bare materials that make up the DNA and are used by the Taq Polymerase to create the DNA fragments.
The Process Overall
When thermal cycling is begun, the thermal cycler will be heated to 95 degrees Celsius in order to reach the temperature needed for the denaturation process of DNA. Denaturation of the DNA double helix occurs when the thermal cycler is held at 95 degrees Celsius for 30 seconds as the two strands of the double helix straighten out and separate. Then, the thermal cycler is cooled to 57 degrees Celsius for 30 seconds so that the primers can be bound to their complementary pairs on the template DNA strands to prepare for the next step, in which the complementary DNA fragment is actually created. The next step is extension, which occurs when the thermal cycler is heated to 72 degrees Celsius for 30 seconds, which is when the Taq polymerase finds the primers, binds to those sequences, and begins to use dNTPs to create complementary base pairs that add to the new desired complementary DNA fragments. The three steps of denaturation, annealing, and extension are repeated until many copies of the target DNA fragment sequence have been made for analysis and experimental use. After the number of cycles desired have been completed, hold the thermal cycler at 72 degrees Celsius for two minutes and then at 4 degrees Celsius. This will stop the chain reaction and make the target DNA fragments stable and ready for future use.
Base Pairing
The process of base pairing through hydrogen bonding allows for the binding of dNTPs to create complementary DNA strand for use in general replication and in PCR. There are four nitrogen bases that are the nucleotides that form DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). Base pairing occurs when these nucleotide bases match to their complements. The complements are adenine to thymine, and cytosine to guanine.
DNA Preparation
After the extraction of the DNA into the PCR tube, it needs to have a couple materials added to it before put it through the replication process. You will need to add two primers (primer 1 and primer 2). These will provide the starting points for replication to occur once the DNA denatures. You will also need to load an assortment of nucleotides. Each of the nucleotides (A,G,C, and T) will be used as building blocks for the complementary DNA replicated. This occurs through base pairing, in which adenine (A) binds to thymine (T), and cytosine (C) binds to guanine (G). and The only remaining substance to be added is the DNA Taq polymerase enzyme. The enzyme will catalyze the process for the actual construction and binding of said building blocks. After these are added, the DNA will be ready to be placed in the thermal cycler.
Thermocycling
After the initial heating of the thermal cycler (to 95°C), the PCR tube loaded with the DNA and materials can be placed inside.
Denaturing the DNA
The act of letting the PCR tube heat up inside the thermal cycler to 95 degrees Celsius (for 30 seconds), is to allow the double helix of DNA spread out and separate into two single strands, thus denaturing. This provides a synthetic opportunity to allow for replication to occur. The temperature sensitive nature of the materials present in the PCR tube prevent primer binding and replication to occur at 95 degrees Celsius, so the PCR tube will need to be cooled in order to move on in the process of PCR.
Allowing the DNA to Anneal
The thermal cycler will need to be set to cool down to 57°C (for another 30 seconds). While this temperature isn't ideal for the entire process of the replication of the DNA, it gives the primers a chance to bind and settle in, as the separated DNA strands will be moving slower at a cooler temperature and more stable. The primers bind to complementary sites on the template DNA through base pairing, marking where the DNA Taq polymerase will bind and start adding nucleotides to create the complementary DNA fragment, but for that to occur at a fast rate, the PCR tube will need to be heated up again in the cycler.
Extending the DNA
The temperature will once again raised, but now to a temperature of 72°C for 30 seconds. This temperature will allow for the DNA Taq polymerase to base pair nucleotides with the template DNA at an optimal rate. After the DNA polymerase does its work, two complementary DNA fragments to the template DNA are created, and as the process is repeated, the target sequence within these fragments will be amplified.
Post
After running through this process a reasonable amount of times, the product is a large amount of desired DNA target sequences equal to two to the power of the number of cycles the polymerase chain reaction was run. To finalize this and stabilize the fragments for future use, cool the PCR tube down to 4°C and let it settle. Consider it now prepared for whatever needs to be done with it.
PCR Visual
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP affects homo sapiens and is pathogenic which means that it can be caused by bacteria, viruses or other kinds of germs. It is located on the 19th chromosome at the 44,907,853 base pair.The disease SNP changes a nucleic acid thymine to a cytosine. It is located on the gene APOE (Apolipoprotein E) which is known as a missense functional consequence and is an important protein for catabolism. It is directly linked to Alzheimer's and subarrachnoid hemmorhage.
Primer Design and Testing
The results of the primer test showed that the non-disease primers targeted DNA fragment of chromosome 19:44907834 to 19:44908053, and the primer melting temperatures as well. For the non-disease forward primer, the melting temperature was 72.7 degrees Celsius, and for the non-disease reverse primer, the melting temperature was 65.3 degrees Celsius. The results of the primer test also showed that there were no matches for the disease specific primers that the group designed, as expected.
BME 100 Fall 2017
