Reza Shahsiah, MD, Pathologist

School of Medicine

Tehran University of Medical Sciences

Polymerase Chain Reaction

History

The Polymerase Chain Reaction (PCR) was

not a discovery, but rather an invention

A special DNA polymerase (Taq) is used to

make many copies of a short length of DNA

(100-10,000 bp) defined by primers

Kary Mullis, the inventor of PCR, was

awarded the 1993 Nobel Prize in Chemistry

Extraction

Optimized buffers lyse samples, stabilize nucleic acids, and enhance selective DNA adsorption to the silica membrane. Alcohol is added and lysates loaded onto the column. Wash buffers are used to remove impurities and pure, ready-to-use DNA is then eluted in water or low-salt buffer. The entire process requires only 20 minutes of handling time (lysis times differ according to the sample source).

How PCR Works

PCR is an artificial way of doing DNA replication

Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times

As in replication, PCR involves:

◦ Melting DNA

◦ Priming

◦ Polymerization

Components of a PCR Reaction

Buffer (containing Mg++)

Template DNA

2 Primers that flank the fragment of DNA to be amplified

dNTPs

Taq DNA Polymerase (or another thermally stable DNA polymerase)

PCR

Melting

94 oC

Melting

94 oC Annealing

Primers

50 oC

Extension

72 oC

Tem

per

atu

re

100

0

50

T i m e

30x

Thermal cycler

Agarose Gel

Real-time PCR

Real-time PCR monitors the fluorescence

emitted during the reaction as an indicator of

amplicon production at each PCR cycle (in real

time) as opposed to the endpoint detection

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Real-Time Principles

Three general methods for the

quantitative assays:

◦ DNA-binding agents (SYBR Green)

◦ Hydrolysis probes (TaqMan)

◦ Hybridizing probes

When to Choose SYBR Green

Assays that do not require specificity of

probe based assays.

General screening of transcripts prior to

moving to probe based assays

When the PCR system is fully optimized -

no primer dimers or non-specific

amplicons, e.g. from genomic DNA

RT detection methods

Fluorescence quenching : the fluorescence of a fluorescent molecule can be quenched by close proximity to a quenching agent upon removal of the quencher, the fluorescence siganl returns

Exonuclease activity: DNA polymerase ability to synthesize new DNA strand ability to remove nucleotides from a second strand of DNA as it's moving on the template

RT detection methods: FRET

RT detection methods: Beacon

Real-time PCR Kinetic

Quantification

Multiplex Real-time PCR

Multiplexing

External Controls

Reagent Blanks (Nontemplate Controls): Applicable reagent controls should be interspersed within each amplification batch run. These controls contain all of the necessary components of the reaction without the addition of template nucleic acid.

Negative Controls Negative controls should contain known nontarget nucleic acid rather than only water or buffer. Always dispense and transfer reagents to negative controls last so that they reflect cumulative effects during manipulations.

External Controls

Positive Controls: A positive

control that has a low

concentration of target

nucleic acid and amplifies

weakly, but consistently

should be selected.

Internal Control

An advantage of using an internal calibrator is that it allows for, but generally does not distinguish between, detection of inhibitors and recognition of nucleic loss during extraction. When added prior to specimen extraction, internal calibrators will undergo the same assay process as the specimen itself.