Mol Bio: Basic PCR Procedure

Polymerase Chain Reaction (PCR)

The primary method for amplifying DNA.

Target Amplification

Copies the target nucleic acid sequence.

Probe Amplification

Amplifies probes specific to the target.

Signal Amplification

Amplifies the detection signal, not the target itself.

PCR

Target Amplification

LCR

Probe Amplification

Branched DNA

Signal Amplification

Hybrid Capture

Signal Amplification

Target Amplification

Involves making copies of a target sequence to such a level (in the millions of copies) that they can be detected in vitro.

Kary Mullis

He conceived the idea of amplifying DNA in vitro in 1983.

Short fragment of the plasmid, pBR322

First successful amplification

Polymerase-catalyzed chain reaction

Kary Mullis called it as

Denaturation

Temperature: 94–96C

Denaturation

Duration: Several seconds to several minutes, depending on the template size.

Denaturation

To separate double-stranded DNA into two single strands for replication.

Annealing

Temperature: 50-70C

Annealing

Crucial for PCR specificity, determining the amplified DNA region.

Annealing

determines amplified DNA region

Annealing

It is affected by factors like primer melting temperature, reaction conditions, salt concentration, mismatches, template conditions, and secondary structure.

Extension

Temperature: 68–72C

Primer Extension Step

DNA synthesis occurs when DNA polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers.

DNA Polymerase Function

Catalyzes the formation of the phosphodiester bond between an incoming dNTP and the base at the 3′ end of the primer.

PCR

duplicates DNA in vitro using DNA polymerase, primer, and template DNA, similar to in vivo replication.

PCR

can amplify specific DNA targets, enabling further analysis and cloning.

PCR

can produce millions of DNA copies (amplicons).

PCR

significantly faster than in vivo replication.

Amplification program (with specified number of cycles and divided into steps).

The components of the PCR are subjected to

Modifications or alternatives to PCR

Other amplification methods are

Generates enough copies of a single gene sequence for analysis.

Purpose of Amplification

Increases the yield of specific nucleic acid sequences for further study.

Purpose of Amplification