PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2, 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
OpenPCR machine: shared by two groups
PCR Reaction Sample List
PCR Reaction Sample
Patient 1, replicate 1
Patient 1, replicate 2
Patient 1, replicate 3
Patient 2, replicate 1
Patient 2, replicate 2
Patient 2, replicate 3
DNA Sample Set-up Procedure
The first step in the DNA sample setup is to take the extracted DNA and put a sample of it into the PCR tube. After this is complete, a sample of Primer 1 will then be taken from its capsule and put into the PCR tube along with the DNA sample. Now, Primer 2 will be added to the PCR tube which will eventually attach into the second site. Next, an excess amount of nucleotides will be added to the PCR tube in order to continue building DNA strands. The final component that will be added is the DNA Polymerase, an enzyme that will build back the strands of DNA using the surrounding nucleotides. Finally, the PCR tube will be placed into a DNA Thermal Cycler, which is a machine able to control the temperature of the PCR environment and dictate key changes in the reaction.
1) Set the DNA Thermal Cycler to 100°C
2) The next step is to set the DNA Thermal Cycler to 95°C and let it run for 2 minutes
3) This process takes approximately 25 cycles which consists of a three step process
The DNA is denatured at 95°C, which divides the DNA Helix into two single strands, for 30 seconds
The DNA is then annealed at 57°C for 30 seconds, which is when the primers attach to the target sequence
The Sample is then heated up until 72°C for 30 seconds, which is when the DNA Polymerase is activated and thus builds the other half of the DNA
4) The sample is then kept at 72°C for 2 minutes
5) Finally, the sample is kept at 4°C
Research and Development
PCR - The Underlying Technology
During the PCR reaction, template DNA functions when the strands are separated, each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. Then primers bind to DNA and serve as a base for the taq polymerase. Taq Polymerase is an enzyme that copies DNA. It is isolated from a heat-loving bacterium that is naturally found in hot springs, so the enzyme doesn't break down at the high temperatures necessary for copying DNA using a PCR. Deoxyribonucleotides (dNTP’s) are the four nucleotides used by DNA polymerase to extend an annealed primer.
Thermal Cycling Process
During the initial step of thermal cycling, the temperature is raised to 95°C and kept there for three minutes. This unwinds the DNA. During the denature step the temperature is kept at 95°C for an additional 30 seconds which leads to the DNA separating into two separate strands. During the anneal step the temperature is dropped down to 57°C for 30 seconds and primers bind to complementary matches on the target sequence. During the extend step, the temperature is raised slightly to 72°C for 30 seconds and DNA Taq Polymerase attaches to the primers and is activated. During the final step, the temperature is kept at 72°C for 3 more minutes allowing the DNA Taq Polymerase to add complementary nucleotides onto the strand in order to create the entire complementary sequence. The cycle repeats multiple times before reaching the final hold step which involves keeping the temperature at 4°C which stops the replication process.
DNA is made up of nucleotides that bind together in a specific way. These base pairs stick together due to hydrogen bonding. Nucleotides adenine and thymine anneal, and cytosine binds to guanine.
Base Pairing Occurrence
Base-pairing occurs during the anneal and final stage. In the anneal stage, primers bind to specific locations on the target sequence to prevent the two template strands from recombining. In the final stage, DNA taq polymerase pulls nucleotides that were previously added to the mixture and attaches them to their complementary base pair until the end of the target sequence is reached.
SNP Information & Primer Design
Background: About the Disease SNP
A single nucleotide polymorphism (SNP) is a DNA sequence variation in one base pair which does not normally occur in other members of the same species. The specific SNP of interest for this laboratory has a reference code rs121908757, and specifically points to a SNP where an adenine nucleotide, in most of the species, becomes a cytosine nucleotide. This small and seemingly minute change in the genome pathogenic and affects the homo sapiens species by greatly increasing the chance of developing the disease cystic fibrosis. This genetic order most commonly causes damage to the lungs and also the digestive system. This SNP occurs on chromosome seven, results in approximately 70% of cystic fibrosis cases.
Primer Design and Testing
Primer and Design Testing Toolbar Results
Primer design consists of being able to find the forward primer and the reverse primer reading them from 5’ to 3’. The non disease forward primer is 19 base pairs in front of where the error in the code is for cystic fibrosis and that one base pair making it 20 total base pairs for the forward primer. The non disease reverse primer starts exactly 200 base pairs in front of the base pair mutation point for cystic fibrosis. Of course this base pair is not mutated in the non disease primers but it is used as a reference point. After adding 200 to the numerical position of the SNP the reverse primer can then be found. In our case the forward primer ends at position 117587799 and the reverse primer ends at 117587999. At that final position we can read from 5’ to 3’, 20 base pairs and obtain the non disease reverse primer. When these primers were plugged into the the genome site, it was successful and these primers were found to be on chromosome 7, and 220 base pairs long. When the disease mutation was used, changing the CGT to AGT, the primers were not found probably because this is a mutation and it is not expected to be on a healthy human genome.