Lecture 5: Polymerase Chain Reaction (PCR) Overview

Polymerase Chain Reaction (PCR)

An in vitro technique that allows the amplification of a DNA fragment from a DNA template.

DNA polymerase

An enzyme that catalyzes DNA synthesis by sequential addition of deoxyribonucleotides to the 3ʹ-OH end of a polynucleotide chain.

Denaturation

The first step in PCR where the double-stranded DNA is heated to separate it into two single strands.

Annealing

The second step in PCR where primers bind to the single-stranded DNA template.

Elongation

The third step in PCR where DNA polymerase synthesizes a new DNA strand complementary to the template strand.

extension

the fourth step in PCR where the primer sequences from their 3' ends to synthesize the target DNA fragment

Cycling

The repeated series of denaturation, annealing, and elongation steps in PCR.

Applications of PCR in Research

Detection of specific genes, sequences, and species identification.

Applications of PCR in Medicine

Identification of pathogens, such as viruses.

Applications of PCR in Biotechnology

Use in recombinant systems.

Applications of PCR in Forensics

Used for paternity tests and other forensic analyses.

3ʹ-OH end

The end of the primer strand to which deoxyribonucleotides are added during DNA synthesis.

Template strand

The strand of DNA that provides the sequence for the synthesis of a new complementary strand.

Primer strand

The short strand of nucleotides that is complementary to the template strand and initiates DNA synthesis.

In vitro technique

A procedure performed outside of a living organism, often in a controlled laboratory environment.

Recombinant systems

Biotechnological systems that involve combining DNA from different sources.

Pathogen identification

The process of detecting infectious agents, such as viruses, using PCR.

Electrophoresis

A technique for separating DNA fragments based on size.

Reaction mix

The combination of components needed for PCR, including DNA template, DNA polymerase, and buffer.

DNA template

The source of DNA to be amplified, which can be genomic DNA, cDNA, plasmid DNA, or a library.

DNA polymerase reaction buffer

A solution that provides the necessary environment for DNA polymerase activity.

Thermostable enzyme

An enzyme that remains active at high temperatures, essential for PCR. i.e. Taq polymerase

Pfu Polymerase

A DNA polymerase derived from Pyrococcus furiosus, known for its proofreading activity.

Proof reading activity

The ability of some DNA polymerases to correct errors during DNA synthesis, specifically in the 3' to 5' direction.

Processivity

The ability of DNA polymerase to continuously synthesize DNA without dissociating from the template.

Fidelity

The accuracy of DNA replication, often higher in polymerases with proofreading capabilities.

Mg2+ [MgCl2]

A cofactor required for the activity of DNA polymerase in PCR.

dNTPs [dATP, dCTP, dGTP, dTTP]

The building blocks of DNA, necessary for DNA synthesis during PCR.

2x Primers (Forward and Reverse)

Short sequences of nucleotides that initiate DNA synthesis during PCR.

H2O

Water, used as a solvent in the PCR reaction mix.

Pipettes / tips

Tools used to accurately measure and transfer small volumes of liquids.

Microtubes & PCR tubes

Containers used to hold samples during PCR and other laboratory procedures.

PCR Instrument

A machine used to perform PCR, providing controlled temperature cycles.