BME100 s2015:Group5 12pmL4
Lab Part A
Section 1: Protocol
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL containing Taq DNA polymerase, MgCl2, and dNTP's
- DNA/primer mix, 50 μl each: all have the same forward and reverse primer but different templates of DNA
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-‐use disposable pipette tips or samples will be cross-‐contaminated
- Cup for discarded tips
- OpenPCR machine
DNA Sample Set-up Procedure
- Obtain patient samples, including a positive and negative control, from the TA as well as the patient ID's for group
- Record which ID's went with each patient and label each sample with the ID consistent with the patient
- Do not write the ID's on the tubes, only record on the table
- Add the polymerase, nDTP, and primers to each of the 8 samples
- The samples are prepared for PCR
Section 2: Research, Development, and the Underlying Technology
Each component of PCR serves a functional component:
- Template DNA is a guide to extend to the primer, which produces new double stranded DNA.
- Primers copy DNA sequences by attaching to either end of the strand, and allow polymerases to start their DNA replication duties.
- Taq Polymeraseis the enzyme responsible for utilizing the surrounding nucleotides to synthesize new strands of DNA based on the template DNA strand
- Deoxyribonucleotides (dNTP's)are nucleotides that are bonded to deoxyribose, which is itself bonded to a phosphate. These provide the structural building blocks for DNA replication.
Knowing the components of PCR is certainly important, but it is more crucial to know what happens to the components during each cycle of thermal cycling in PCR.
- INITIAL STEP: 95°C for 3 minutes: Bringing the temperature up to 95 degrees Celsius causes all the DNA samples in the PCR machine to start to unwind
- Denature at 95°C for 30 seconds: As the double helix structures start to unwind, there will be twice as many single stranded DNA than the original amount, altering the DNA in this way is referred to as denaturing
- Anneal at 57°C for 30 seconds: When the DNA is cooled, the single stranded DNA will want to combine back into its original state, but by this time, the primers have already attached to the target DNA, which have already become a double helix and will leave the original strands to still be single stranded. At this point the DNA will separate again, leaving twice as many single strands of target DNA compared to the original DNA.
- Extend at 72°C for 30 seconds: At this temperature, the polymerase is able to come into play. The polymerase will attach itself to the target DNA and create the appropriate base pairs for the DNA, stopping at the primers attached to the ends of the target DNA. This yields a fully reconstructed double helix of the target DNA.
- FINAL STEP:72°C for 3 minutes: This step is in place to ensure that the single stranded DNA is fully extended
Each of the nucleic bases pair with another base.
- Adenine (A) pairs with Thymine (T)
- Thymine (T) pairs with Adenine (A)
- Cytosine (C) pairs with Guanine (G)
- Guanine (G) pairs with Cytosine (C)
Base pairing occurs between the steps of Annealing and Extension steps. During annealing, the system is cooled and the target DNA is binded with primers that serve as starting positions for replication. The temperature is slightly elevated the thermal cycling stage of extension occurs, which is where two taq polymerase comes in and matches the base primers with their pairs and extend the primers forming new nucleotide strands of desired target sequences of DNA. These two steps combined signify the completion of the base pairing.