Polymerase Chain Reaction (PCR) Overview

Polymerase Chain Reaction (PCR) Overview

  • Definition of PCR:

    • PCR is a technique for amplifying a specific sequence of DNA.

    • Applications include:

    • DNA fingerprinting

    • Detecting viruses or bacteria (e.g., COVID PCR tests)

    • Diagnosing genetic disorders

Reagents Used in PCR

  • DNA:

    • Isolated pGLO plasmids are used to amplify the pBAD promoter of the Green Fluorescent Protein (GFP) region.

  • DNA Primers:

    • Primers are short DNA sequences (18-20 base pairs in length) that bind to either end of the target DNA sequence.

  • PCR Beads:

    • Contain Taq Polymerase, an enzyme that synthesizes new strands of DNA.

  • Nucleotides (dNTPs):

    • Required for building DNA strands, consisting of adenine (A), thymine (T), cytosine (C), and guanine (G).

  • Water:

    • Used to dilute and mix reagents appropriately.

Functionality of pBAD Promoter and GFP

  • Role of Promoters:

    • Promoters initiate the transcription of genes by copying DNA into RNA.

  • Transcription Necessity:

    • Transcription is essential for the breakdown of arabinose involving digestive enzymes (araA, araB, and araD).

  • AraC Protein Function:

    • AraC protein controls transcription:

    • Prevents or initiates transcription based on the presence of arabinose.

  • Modification in pGLO Plasmid:

    • The pGLO plasmid replaces the genes araA, araB, and araD with the gene for GFP.

  • Arabinose Presence:

    • When arabinose is present, AraC activates the pBAD promoter, leading to the production of GFP.

PCR Process Overview

  • General Process of PCR:

    • After reagents are mixed and added to PCR tubes, they are placed in a thermocycler.

    • The thermocycler alters the temperature and duration of heating and cooling to facilitate DNA strand synthesis.

  • Three Main Steps of PCR:

    1. **Denaturing

      • Temperature:** 95℃

      • Function:** High temperature breaks the hydrogen bonds of double-stranded DNA, resulting in two single DNA strands.

    2. **Annealing

      • Temperature:** 55℃

      • Function:** Allows primers to bind to their respective ends of the single DNA strands.

    3. **Elongation/Extension

      • Temperature:** 72℃

      • Function:** Taq Polymerase synthesizes new DNA strands by adding nucleotides to the available single strands.

Amplification Iteration

  • Cycle Completion:

    • After the three steps, two copies of the desired DNA sequence form.

    • This cycle repeats 29 additional times, resulting in millions of copies of the pBAD DNA sequence.

Detailed PCR Protocol

  • PCR Cycle Details:

    1. Initial Denaturing:

      • At 95℃ for 3 minutes.

    2. Denaturing Cycle:

      • At 95℃ for 30 seconds.

    3. Annealing Cycle:

      • At 55℃ for 30 seconds.

    4. Elongation Cycle:

      • At 72℃ for 1 minute.

    • Steps 2-4 are repeated 29 times for a total of 30 cycles.

    1. Final Elongation:

      • At 72℃ for 5 minutes.

    2. Hold:

      • At 4℃ (infinite hold, program ends).

Reagent Addition Procedure

  • Order of Addition:

    • Reagents should be added from least important to most important.

    • Example volumes to add to PCR tubes:

    • ___ µl Water

    • 2 µl Forward Primer

    • 2 µl Reverse Primer

    • ___ µl DNA (10ng of your +pGLO Plasmid Isolate)

    • Total Volume: 25 µl

    • Calculate the volume of DNA needed to obtain 10ng, adjust the amount of water accordingly to achieve the total volume of 25µl.

    • Use a centrifuge to spin PCR beads; ensure they are labeled with group initials and date to avoid confusion.