PCR and Electrophoresis Techniques

• These notes focus on the application of PCR and electrophoresis techniques to the study of nucleic acids, which is relevant to molecular biology and cytogenetics
• The content covers PCR principles, components, design, and variations. It also addresses electrophoresis of DNA

Introduction

• Polymerase Chain Reaction (PCR) is a technology that allows the in vitro replication of a DNA molecule in a selective manner
• PCR involves amplification of a target sequence to obtain an amplified product
• This product can be used for identification or quantification

Fundamentals of PCR

• PCR requires a DNA sample in addition to specific conditions to amplify a desired sequence
• Primers are specific sequences designed to initiate DNA polymerase activity
• This process generates millions of copies of the sequence of interest
• Electrophoresis involves techniques that separate molecules of nucleic acids and proteins for identification
• Electrophoresis is sometimes used for the separation of these molecules; both processes are based on the movement of molecules in an electric field

Polymerase Chain Reaction (PCR)

• This technique duplicates DNA, replicating under controlled in vitro conditions to amplify a specific sequence from a DNA sample
• Specificity is a key feature achieved through well-designed primers

Primers

• Primers are short DNA sequences that attach to specific regions
• Primers enable the amplification of sequences that include the sequence that acts as the start for DNA-polymerase

Molecular Cloning

• PCR is considered a molecular cloning technique
• PCR produces multiple copies of a particular sequence

PCR History

• Prior to PCR, replication required in vivo procedures like recombinant DNA experiments which were inefficient
• Kjell Kleppe proposed a procedure in 1971, though it was not well-recognized
• Kary Mullis developed the idea in 1983 and is credited as the discoverer of PCR
• Kary Mullis won Nobel Prize in Chemistry for studies in directed mutagenesis

PCR Components

• PCR requires multiple components, each with important functions
• Primer design is essential, and the primers' must correspond to region of sequence
• Oligonucleotides of single-stranded DNA, 18-24 bases, must be complementary to target region
• Paired primers are called “forward” and “reverse”, binding to the 5' and 3' ends, respectively

Deoxynucleotide Triphosphates (dNTPs)

• dNTPs are essential for DNA synthesis in PCR
• dNTPs include building blocks: adenine, guanine, cytosine and thymine (dATP, dGTP, dCTP, dTTP)
• Must also be added in excess to avoid shortages to ensure proper results with concentrations between (20-200 μΜ)

DNA Polymerase

• DNA polymerase is used to catalyse DNA duplication
• The polymerase must have the ability to avoid degradation at extreme temperatures
• The most common polymerase is Taq-polymerase, coming from Thermus aquaticus

Thermo-Stable Enzymes for PCR

• Tth, from Thermus thermophilus and Pfu (from Pyrococcis furiosus), Vent (Thermococcus litoralis) are other enzymes used in PCR
• The latter two enzymes have proofreading capabilities (therefore: high-fidelity)

DNA Template

• Template DNA has a sequence being amplified that is purified for a specific sequence
• Template DNA must be extracted in purified format

Buffer Solutions

• These solutions are used to regulate pH
• Buffer solutions are is aqueous and contain KCI and Tris

Magnesium Chloride

• Acts like a cofactor of polymerase
• Included in the reaction's buffer solution

"Mastermix"

• Commercial kits contain all PCR components except the DNA template in a pre-mixed solution called Mastermix

Primer Design

• Primer design must meet specification for effective PCR
• Sequencing techniques and bioinformatics tools aid designs

Primer Length

• Primers should contain 18 nucleotides, at minimum, to be effective and not create any risk

Optimal Hybridization Temperature

• Hybridization temperature should be as close as possible to melting point of the sequence
• An ideal hybridization temperature should be above 45°C
• Hybridization temperature can be estimated using the formula: Ta= 4 (G+C) + 2 (A+T)

Presence of Homopolymers

• Homopolymers, including guanine, should be avoided to prevent polymerase slippage and signal reduction, particularly in quantitative PCR

Single Nucleotide Polymorphism

• Primers' need to be designed so single single nucleotide polymorphisms. Single Nucleotide Polymorphism (SNP) regions should be avoided

GC Content

• The GC content of primers should ideally be between 40-60% to ensure stable hybridization without affecting polymerase function

Autocomplementarity

• Primers should lack complementary or palindromic sequences to prevent primer dimers or hairpin structures, which would inhibit hybridization

Purity

• Primers must be purified for PCR for the most effective result
• The HLPC technique provides very purifed primers