Polymerase Chain Reaction

PCR

Polymerase Chain Reaction (PCR) is a technique that allows the rapid amplification of a specific DNA region in vitro. The basis of this method is the ability of DNA polymerase to synthesise new strands of DNA, complementary to a given template strand. The primary components of this technique are: (i) DNA template, (ii) DNA polymerase (TaqDNA Polymerase is the most commonly used enzyme), (iii) heat-resistant Primers (short, single-stranded DNA – two primers are used in each PCR reaction such that they flank the target region), for DNA synthesis, and (iv) Nucleotides. The aim of PCR is typically to provide enough copies of a target DNA region to allow for further analysis – for example, gel electrophoresis allows the visualisation of PCR results.

The system being utilised will influence the optimal operating conditions for the concentration of Taq DNA Polymerase, template DNA, primers and MgCl2 (required for DNA Polymerase functioning). TaqDNA Polymerase and MgCl2 can be titrated to determine the optimum efficiency. See below for a general protocol for a 50 µl PCR, with recommendations.

A. Reagents

  • 5 µl 10X Standard Taq Reaction buffer
  • 1 µl 10 mM dNTPs
  • 1 µl 10 mM Forward Primer
  • 1 µl 10 mM Reverse Primer
  • Template DNA
  • 25 µl Taq DNA Polymerase
  • Make up to 50 µl Nuclease-free water

B. Procedure

The protocol is composed of 3 basic steps: denaturation, annealing, and extension.

  1. Preheat the thermocycler to 95oC.
  2. Gently mix the reaction components. If necessary, centrifuge briefly to collect the liquid to the bottom of the tube.
  3. If using a PCR machine without a heated lid, overlay the sample with mineral oil.
  4. Transfer PCR tubes from ice to the preheated PCR machine (95oC) for the initial denaturation step.
  5. Begin thermocycling:
    • Initial denaturation: 95 °C, 30 seconds
    • 30 cycles of: 95 °C, 15-30 seconds; 45 °C – 68 °C, 15 – 60 seconds; 68 °C, 1 minute/kb
    • Final extension: 68 °C, 5 minutes
    • Hold: 4 – 10 °C
  6. Validate the final reaction mixture by running a fraction on an agarose gel with an appropriate molecular marker. Directly sequence the amplified product or carry out restriction enzyme digests.

Recommendations

  • MgCl2: For most Taq DNA Polymerase a MgCl2concentration of 1.5 – 2.0 mM is optimal. The final concentration in standard 1X Standard Taq Reaction Buffer is 1.5 mM. If another concentration is optimal, a reaction buffer that does not contain MgCl2 can be used and the MgCl2 can be added separately.
  • Additives: DMSO and formamide can be added to improve difficult targets such as GC-rich sequences.
  • Template DNA: Use high quality, purified DNA. For a 50 µl reaction, recommended amounts are:
    • Genomic DNA 1 ng – 1 µg
    • Plasmid or viral DNA 1 pg – 1 ng
  • Taq DNA Polymerase: 1.25 units/50 µl reaction is recommended, however this can vary from 0.25 – 2.5 units/50 µl reaction.
  • Denaturation: A longer initial denaturation step (2 – 4 minutes at 95 °C) can be employed for difficult templates such as GC-rich sequences. For colony PCR 5 minutes at 95 °C is recommended. During themocycling 15 – 30 seconds at 95 °C is recommended.
  • Annealing: The temperature is dependent on the TM of the primer pair (usually 45 °C – 68 °C). Optimise by carrying out temperature gradient PCR starting 5 °C below the TM.
  • Extension: The recommended temperature is 68 °C with the time generally 1 minute per kb. A final extension of 5 minutes at 68 °C is recommended.
  • Cycle number: Generally, 25 – 35 cycles yields sufficient product. Up to 45 cycles can be employed to detect low copy number targets.