LAB 4 WRITE-UP
- Lab coat and disposable gloves
- 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
- 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
- Label PCR tubes as positive, negative, patient 1 (replicant 1,2,3), and patient 2 (replicant 1,2,3) then place in rack
- Add 50 microliters of the PCR reaction mix with pipette to each tube and then discard disposable pipette
- Add 50 microliters of the DNA mix to each tube with NEW pipette, remembering to use the specific DNA mix for each labeled tube, and then discard disposable pipette
- Close tubes and place them in thermal cycler
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
Research and Development
PCR - The Underlying Technology
DNA and PCR components
DNA is known as the blueprint of life and the components that make up DNA are called nucleotides. The four nucleotides that form DNA are Thymine, Cytosine, Adenine, Guanine. PCR, polymerase chain reaction, is a method of analyzing DNA. There are a couple different components involved in PCR and each component has its own purpose. The template DNA is the section of DNA that PCR will target to amplify. The next component are primers which give a starting point for the replication of each strand of the template DNA. The primers for this lab are 20 bases long and are essential for the next component, Taq polymerase. Taq polymerase attaches to the primer and replicates DNA by combining base pairs of each strand of DNA. The usage of Taq polymerase is useful because it can withstand the high temperature that is required to denature DNA into straight strands. The last component is the dNTPs or deoxyribonucleotides which are spare nucleotides that the Taq polymerase uses to make the base pairs in the replication of DNA.
Thermal Cycling in PCR
The process of PCR is very sensitive to temperature. The beginning of the thermal cycle begins when the cycler is heated to 95 degrees Celsius for 30 seconds. At this temperature, DNA is denatured and instead of being in a double helix shape, the DNA is changed to a linear shape as the two strands straighten out. The cycler is then cooled to 57 degrees C for 30 seconds as the primers attach themselves to the separated strands of DNA. The temperature is again changed to 72 degrees C for 30 seconds as the Taq polymerase binds to the primers and uses the dNTPs to create the base pairs and DNA is replicated. This cycle is repeated 25 times until the final step of holding 72 degrees C for 2 minutes until the final cooling temperature of 4 degrees C.
If there is a variation in the way these nucleotides are arranged, it is known as polymorphism. A specific type of polymorphism is SNP or single nucleotide polymorphism where only a single nucleotide is different and causes the variance. The SNP that is analyzed for the lab is “rs769452” which is a variation found in Homo sapiens and its chromosome location is 19:44907853. This SNP has high clinical significance as it is pathogenic and is linked with Alzheimer’s.
The SNP analyzed in the lab is found in the APOE gene. This gene is responsible for giving instruction for making apolipoprotein E. The main functions of this protein is for amyloid-beta binding, antioxidant activity, and cholesterol binding. Alleles are alternative forms of a gene and if a person has a certain allele of the APOE gene, it increases their likelihood of developing Alzheimer’s. The non-disease associated allele has the codon CTG while the disease-associated allele is changed to CCG. The numerical position of this SNP is 44907853. This numerical position will be used to find the location of the forward and reverse primer for polymerase chain reaction, or PCR.
Primers used in PCR to target SNP
PCR requires two primers, the forward and reverse primer. The function of these two primers is to target a specific sequence of DNA that the Taq Polymerase can attach to. When designing the forward primer, the sequence is twenty bases long and ends with the nucleotide that causes the variance in the DNA sequence. The non-disease forward primer would be 5’- A G C G G C C A G C G C T G G G A A C T. Due to the SNP, the disease forward primer be 5’-A G C G G C C A G C G C T G G G A A C C. The only difference between the two forward primers is the very last nucleotide which is changed from T to C because of the SNP. The reverse primer’s starting position is 200 bases to the right of the disease SNP which the numerical position is 44908053 on the bottom strand. When making this primer, it is written in reverse order as the the first base is in the 44908053th position and the following bases are toward the SNP towards the left. There is no difference between the non-disease and disease reverse primer because there is no SNP in this portion of the DNA sequence.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP stands for single nucleotide polymorphism and is basically the exchange of a single nucleotide which means the arise of an additional allele. Some exchanges of a single nucleotide do not affect the translation/transcription at all since it can be a non-coding part of the genome or the DNA-polymerase might transcript the other strand of the DNA which would not result in a disease. This change can also lead to major complications once translated and transcripted where the protein produced is a completely different one to the original. Several very serious diseases are correlated with SNP such as Alzheimer’s, cystic fibrosis and sickle-cell anemia. Looking at SNP is what lets scientists predict whether or not an individual is predispositioned to develop a disease.
Primer Design and Testing
A test was conducted to observe the impact on the DNA transcription and protein translation when one codon is changed by one nucleotide. The codon that was manipulated was on the 19th chromosome in the numerical position of 44907853. The change of the nucleotide resulted in an allele producing the protein called Apolipoprotein E. The first screen capture shows a search for the gene in its non-manipulated form and shows that it does exist in the human genome. The second screen capture shows the manipulated form and no matches were found just as can be expected. The web page used only has the non-manipulated genes in its system which is why the SNP could not be found.