Molbio: PCR Modifications

The Multiplex PCR

Detects multiple targets in a single PCR run

Reverse Transcriptase PCR

Uses RNA templates

Nested PCR

Uses amplified products as templates

Quantitative PCR

Quantitates starting template

Multiplex PCR

Used for typing or identification analyses, pathogen typing, forensic identification, and detecting multiple organisms in clinical microbiology.

Multiplex PCR

Allows simultaneous amplification of multiple products

Multiplex PCR

Requires complex optimization due to potential amplification efficiency differences and primer interference, making it difficult to achieve optimal amplification for all products.

Reverse Transcriptase PCR (RT-PCR)

converts RNA to double stranded DNA (cDNA) using reverse transcriptase and primers, enabling PCR amplification.

Reverse Transcriptase PCR (RT-PCR)

used for gene expression analysis, detecting RNA viruses, and analyzing genes with long introns.

RT-PCR Modification

The amplification program includes an initial incubation step for RT to synthesize cDNA from RNA

RT-PCR Modification

vulnerable to RNA degradation, requiring careful specimen handling for accurate results.

Nested PCR

A modification of PCR that increases sensitivity and specificity by using two pairs of primers to amplify a single target in two separate PCR runs.

Semi-nested PCR

one of the second-round primers is the same as the first-round primer.

Fluorescing agents

1. EtBr (Ethidium bromide)

2. SYBR green

The Real-Time or Quantitative PCR

Uses fluorescence to detect the growing target copy number during PCR, allowing analysis in the exponential phase of growth.

Real-Time/Quantitative PCR

A modification of the PCR process that allows for the accurate determination of the amount of starting template.

Real-Time or Quantitative PCR

measures target copy number during PCR amplification, resulting in a curve similar to a bacterial growth curve.

Threshold Cycle (CT) in qPCR

is the PCR cycle at which sample fluorescence crosses a threshold, allowing for the determination of the starting amount of an unknown specimen.

Inverse relationship

Fluorescence vs. CT Relationship

Real-Time or Quantitative PCR

analyzes fluorescence during the exponential phase

Endpoint Analysis

measures products at the plateau, where they are all at the same level.