PCR Lecture 13.3.25

The Polymerase Chain Reaction (PCR)

Overview

The Polymerase Chain Reaction (PCR) is a laboratory technique that enables the amplification of a specific region of DNA, allowing for the production of numerous copies from a single template. This method is invaluable in molecular biology and has diverse applications ranging from research to clinical diagnostics.

Lecture Objectives

• Understand the components and their roles in PCR.

• Familiarize with the stages of the PCR reaction and their significance.

• Learn about primer design and how they interact with target sequences.

• Discuss methods for optimizing PCR for effective amplification.

Key Components of PCR

The PCR "Recipe"

1. DNA Template: The starting material for PCR, which contains the target sequence along with a significant quantity of non-specific DNA from the sample.

2. Primers: Short nucleotide sequences that are designed to bind to specific sites flanking the target sequence.

   • Forward Primer: Complementary to the reverse strand and corresponds to the 5' end of the forward strand.

   • Reverse Primer: Complementary to the forward strand; matches the 5' end of the reverse strand.

3. DNA Polymerase: The enzyme responsible for DNA synthesis during PCR. It adds nucleotides to the growing DNA strand.

   • Taq polymerase is commonly used due to its thermostability, which allows it to function at high temperatures necessary for DNA denaturation.

4. Deoxynucleotide Triphosphates (dNTPs): The building blocks of DNA, comprising adenine (dATP), thymine (dTTP), cytosine (dCTP), and guanine (dGTP). These nucleotides are essential for constructing new DNA strands.

5. Buffer: A substance that provides optimal conditions (e.g., pH) for DNA polymerase activity, often containing magnesium ions (Mg2+) which are crucial for enzyme activation and stabilizing the nucleotide structure.

Process of PCR

Stages of PCR

PCR involves multiple cycles, typically around 40, and consists of the following stages:

1. Initial Denaturation: The double-stranded DNA (dsDNA) is separated into single strands by breaking hydrogen bonds, usually at around 95°C.

2. Denaturation: Maintains the separation of dsDNA at high temperatures.

3. Annealing: The temperature is lowered to allow primers to attach to their complementary sequences on the target DNA.

4. Extension: DNA polymerase synthesizes new DNA strands by adding dNTPs in the 5' to 3' direction, using the 3' OH group for nucleotide addition.

5. Final Extension: Any remaining single-stranded regions are extended to ensure complete amplification of the target sequence.

Primer Design

Considerations for Designing Primers

• GC content should be between 40% to 60% for stable binding.

• Inclusion of a GC 'clamp' in the last five bases (3' end) is important for binding specificity.

• Length of primers should be between 18-24 bases.

• Melting temperature (Tm) of primers should be within a 5°C range of each other (ideally between 50-60°C).

• Avoid regions of complementarity within and between primers to prevent undesired secondary structures, such as cross dimers, self dimers, or hairpins.

Melting Temperature (Tm) Calculation

Using the Wallace Rule:

• Tm (°C) = 4 x (G + C) + 2 x (A + T)

Using the Howley Rule:

• Tm (°C) = 81.5 + 16.6 [log10([Na+])] + 0.41 (%G + C) – 600 / nt

Where [Na+] is the sodium concentration and nt is the length of the oligonucleotide.

Optimisation of PCR

Optimizing PCR is crucial for successfully amplifying the target sequence without non-specific amplification. Key parameters to optimize include:

• DNA template concentration

• Buffer concentration (particularly Mg2+)

• Primer concentrations

• Annealing temperatures

• Number of PCR cycles

Assessing PCR Success

• Gel Electrophoresis: Used to visualize PCR products and confirm successful amplification of the target DNA.

• Sanger Sequencing: Provides detailed analysis and verification of the amplified DNA sequence, ensuring correctness of the amplification process.