LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTPs
- 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
| G26 P || Positive Control || None
| G26 N || Negative Control || None
| G26 1-1 || Patient 1, Replicate 1 || 47894
| G26 1-2 || Patient 1, Replicate 2 || 47894
| G26 1-3 || Patient 1, Replicate 3 || 47894
| G26 2-1 || Patient 2, Replicate 1 || 77482
| G26 2-2 || Patient 2, Replicate 2 || 77482
| G26 2-3 || Patient 2, Replicate 3 || 77482
DNA Sample Set-Up Procedure
- Procure all of the materials listed above, including the patient samples and positive and negative control samples.
- Cut the strip of empty PCR tubes into two strips of four linked tubes. This will allow for all of the tubes to fit into the OpenPCR machine.
- Label the sides, NOT the lids, of the empty tubes with a black marker in the following manner, which corresponds to each of the samples: G26 P, G26 N, G26 1-1; so on, so forth.
- Place the tubes in a rack, so that the PCR reaction mix and DNA/primer mix can be transferred into them.
- Use proper pipetting technique to transfer 50 μL of PCR reaction mix into the empty positive control tube. After transferring the PCR reaction mix, be sure to dispose of the pipette's tip into a cup to ensure that the samples do not become cross-contaminated.
- Use a new pipette tip to transfer the DNA/primer mix corresponding to the positive control sample into the same tube, to which the PCR reaction mix was just added - the total volume of the positive control tube (and all of the other tubes in the end) should now be 100 μL.
- Repeat steps 5 and 6 for all of the other samples, making sure to pay attention that the correct DNA/primer mixes are added to their matching tubes.
- Close the lids of the PCR reaction tubes and ensure that they are securely closed.
- Bring the tubes over to the OpenPCR machine and place them into slots in the heating block. It is important to start the machine only when all of the slots have been filled.
The thermal cycling program is a process used in PCR, through which DNA is heated and cooled in a systematic manner to allow the DNA to go through denaturation, annealing, and extending - and thus be amplified through the PCR process.
Number of Cycles: 35
- Heated Lid: 100°C
- Initial Step: 95°C for 2 minutes
- Denature: 95°C for 30 seconds
- Anneal: 57°C for 30 seconds
- Extend: 72°C for 30 seconds
- Final Step: 72°C for 2 minutes
- Final Hold: 4°C
Research and Development
PCR - The Underlying Technology
What Is the Function of Each Component Involved in a PCR Reaction?
- The template DNA is the pre-existing DNA sample that contains the target sequence, allows primers to bind to the sequence, and allows Taq polymerase to match nucleotides to it, thus amplifying the DNA sample. The primers are short, custom-made pieces of single-stranded DNA synthesized in a laboratory and complementary to the target sequence. Primers allow Taq polymerase to attach to them and to extend the strands to duplicate the target DNA sequence. Taq polymerase is a heat-resistant enzyme that synthesizes new strands of DNA that are complementary to the target sequence. The nucleotides (dNTPs) are single units of the A, T, G, and C DNA bases that are added to the polymerase chain reaction and provide the Taq polymerase building blocks to synthesize DNA strands complementary to the template DNA.
What Happens to the PCR Reaction Components During Each Step of Thermal Cycling?
- At the beginning of PCR, the temperature is set at 95ºC for 2 minutes to allow for the complete denaturation of the template DNA. After this initial step, the actual denaturing step lasts approximately 30 seconds at 95ºC. Denaturing describes the process through which double-stranded DNA separates into single strands, as the near-boiling point temperature disrupts the hydrogen bonds between the nitrogenous bases in the DNA. Next, the temperature is brought down to 57ºC for 30 seconds to allow for two primers to bind, or anneal, onto the target sequence on one of the strands of the template DNA, preventing the strands from pairing back together. The temperature is then brought up to 72ºC for another 30 seconds, which activates the Taq polymerase. Upon locating a primer, the Taq polymerase will attach to the single DNA strand and begin to add complementary deoxyribonucleotides (dNTPs) to the single DNA strand, extending the DNA. The Taq polymerase continues until it gets to the end of the strand and falls off. Then, the temperature is held at 72ºC for 2 minutes to allow Taq polymerase to finish fully extending any remaining single-stranded DNA with dNTPs. Once PCR is complete, the thermal cycler is set to 4ºC to allow for the DNA strands to bind together and to form double helices again. This temperature allows for the samples to be stored until they are ready to be used. The thermal cycling steps of PCR are repeated until a desired amount of copies of the template DNA are created.
Which DNA Bases Anneal to Which Bases?
- DNA is made up of three essential components: a backbone consisting of phosphate groups and deoxyribose sugar, and four types of nitrogenous bases - adenine (A), cytosine (C), guanine (G), and thymine (T). Base-pairing occurs between purines (adenine and guanine) and pyrimidines (thymine and cytosine), which are linked to each other by electrostatic attractions called hydrogen bonds. The nitrogenous bases in DNA anneal to other bases as shown by the list below:
- Adenine (A) anneals to thymine (T)
- Thymine (T) anneals to adenine (A)
- Cytosine (C) anneals to guanine (G)
- Guanine (G) anneals to cytosine (C)
- Thus, in DNA molecules, there are two different types of base pairs - adenine-thymine base pairs and guanine-cytosine base pairs.
When Does Base-Pairing Occur During Thermal Cycling?
- Base-pairing occurs during the annealing and extending steps of thermal cycling. Base-pairing occurs in the annealing step of thermal cycling, as the primers form base pairs with the target DNA sequence by binding adjacently to the complementary nucleotides on the target DNA sequence. In the extending phase of thermal cycling, base-pairing also occurs, as Taq polymerase adds the nucleotides that directly correspond and form base pairs with the bases present on the template DNA strand, which allows for new double strands of DNA to be formed.
BONUS: Original Illustration of PCR
DNA Denaturation at 95º Celsius
Primer Annealing at 57º Celsius
DNA Extension at 72º Celsius
Final Hold at 4º Celsius