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
- Cup for discarded tips
- OpenPCR machine
PCR Reaction Sample List
| Tube Label || PCR Reaction Sample || Patient ID
| G4 + || Positive control || none
| G4 - || Negative control || none
| G4 1-1 || Patient 1, replicate 1 || 86057
| G4 1-2 || Patient 1, replicate 2 || 86057
| G4 1-3 || Patient 1, replicate 3 || 86057
| G4 2-1 || Patient 2, replicate 1 || 31866
| G4 2-2 || Patient 2, replicate 2 || 31866
| G4 2-3 || Patient 2, replicate 3 || 31866
DNA Sample Set-up Procedure
- Step 1: Collect DNA that has been extracted from cells.
- Step 2: Move the DNA into a PCR tube.
- Step 3: Add Primer 1 to the PCR tube which will attach to sites on either end of the DNA strand segment.
- Step 4: Add Primer 2 which will attach to the second site on the DNA.
- Step 5: Add nucleotides to the PCR tube that will be used to create many DNA copies.
- Step 6: Add DNA Polymerase to the PCR tube which is used to read the DNA code and form a new DNA copy.
- Step 7: Place the PCR tube into a DNA 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
Functions of the Components of a PCR Reaction
In a PCR reaction, the template DNA serves as the base strand of DNA of which copies will be made throughout the process. This template DNA, once heated to 95℃, will separate into two single strands. From there, a primer will bind to a certain section of the DNA in order to indicate where on the strand the replication should start. Another primer will bind to the end of a select strand of DNA, indicating the end of the target sequence of DNA. Then, the enzyme Taq polymerase will bind to the start primer and extend it by adding the complementary deoxyribonucleotides (dNTP’s) to a single strand of DNA, thus completing the process of replication.
The Results of the Steps of Thermal Cycling on the Components of a PCR Reaction
At the beginning of the process, the sample with the double-stranded template DNA in it will be heated up to 95℃ for three minutes. This template DNA will then denature at 95℃ for 30 seconds. From there, the solution will be cooled down to 57℃, during which the primers will attach to the newly separated single strands of DNA. Then, when the temperature is increased to 72℃, Taq polymerase will take 30 seconds to bind to the starting primers and begin to “extend” the DNA by adding the complementary deoxyribonucleotides, a process otherwise known as base-pairing. The temperature is then kept at 72℃ to ensure that primer extension is completed. Lastly, the sample is exposed to a cooling process from 72℃ to 4℃ in order to set the DNA and make sure it is firm. This DNA is now ready to be used as template DNA for the next round of replication.
Base Pairing of Nucleotides
A DNA strand is made up of four nucleotides that are assigned with the abbreviations A, T, C, and G. When base pairing occurs Adenine (A) from one single strand of DNA will pair up with Thymine (T) from a different single DNA strand. Likewise, Cytosine (C) from one single DNA strand will pair up with Guanine (G) from another single DNA strand.
Base-pairing During Thermal Cycling
Base-pairing occurs during the Extend step of thermal cycling, in which a single strand of DNA is replicated by Taq polymerase, and at the Final step of thermal cycling because 72°C is the ideal temperature of DNA base-pairing. At this temperature the Taq DNA Polymerase is able to function optimally.
SNP Information & Primer Design
Background: About the Disease SNP
The disease SNP stands for single nucleotide polymorphism. A single nucleotide polymorphism occurs when a single base pair changes within a genome. Naturally SNPs occur once in every 300 nucleotides. However most SNPs do not have an effect on health or development. SNPs can indicate the risk of development of particular diseases, and may help predict an individual's response to certain drugs. More often than not, a single SNP does not affect an individual. Rather, SNP's work in coordination with other SNP's to exhibit an actual disease.
Primer Design and Testing
The forward and reverse primers were found for both the disease and the non-disease variations. The forward primer for the non-disease variant was 5’- C A G C A T C G T G A C C C T G C C G C and the reverse primer was 5’ – C T T G T G G A G C C G G G C G A T G C. The forward primer for the disease variant was 5’ - C A G C A T C G T G A C C C T G C C G T which was identical to the non-disease variant, but with the last nucleic base C switched with a T, and the reverse primer was identical to the non-disease variant. After entering these into the non-disease genome sequencing website provided, the chromosome location result given was: chr16:89986125 for the non-disease variant and no results were returned for the disease variant. The chromosome location according to NCIB was chr16:89919736. It is not clear on what caused this minor difference. No mistake was found during troubleshooting and double checking. It is possible that this was a result of updated information.