Polymerase Chain Reaction (PCR) Overview

Polymerase Chain Reaction (PCR)

• PCR is a method for exponentially amplifying a specific DNA sequence in vitro.
• It can be used to alter or add new sequence information to the amplified sequence.
• Knowing the ends of the sequence is crucial for synthesizing oligonucleotides that hybridize to the ends.
• A small amount of the sequence is needed to initiate the reaction.
• The target sequence doesn't need to be in pure form; it can be part of a larger molecule.
• The reaction produces a discrete double-stranded DNA (dsDNA) molecule with termini corresponding to the 5' ends of the oligonucleotides used.
• PCR can amplify a small segment of a larger molecule to a desired amount using specific oligonucleotides complementary to different strands of the target sequence.

Reaction Cycle

• The process involves cycles of denaturation, annealing, and extension.
• Denaturation: High temperature (e.g., 94-96°C) separates the DNA strands.
• Annealing: Lower temperature allows primers to bind (hybridize) to complementary sequences on target DNA.
• Extension: DNA polymerase extends the primers using nucleotides.

Exponential Amplification

• The process is repeated until the desired amount of the target sequence is produced.
• The product of each cycle acts as a template for the next, resulting in exponential amplification.
• The amount of product is directly proportional to the number of cycles.

Yield per Cycle

• A yield per cycle of roughly 62% was calculated.
• Higher target concentrations might lead to higher yield percentages.

Materials and Methods

• Oligonucleotides are synthesized using automated DNA synthesis machines.
• Deoxyribonucleoside triphosphates and other reagents are used.
• Agarose or polyacrylamide gels are used for DNA analysis.

Polymerase Chain Reaction Methods

• Different methods are described for various target amounts and specific needs.
• Method I: Uses plasmid DNA as a template; specific oligonucleotide pairs.
• Methods II, III, IV, V, VI: Focus on various amounts of the target DNA, using primers, buffers and enzymes.
• Different cycles of denaturation, annealing and extension, as well as different enzymes.

Detection of Minute Quantities of DNA

• The PCR method coupled with labeled hybridization probes can detect single-copy nucleic acid sequences in complex samples.
• It offers a faster and more accurate alternative for analysis and cloning, particularly when dealing with small amounts of target DNA.

In Vitro Mutations

• "Mispriming" can be utilized to make intentional mutations in a sequence or to add sequence information.
• Primers that imperfectly match the target will attach and produce non-targeted sequences that provide variation to the initial sequence.