Reza Shahsiah, MD, Pathologist School of Medicine Tehran ...
Transcript of Reza Shahsiah, MD, Pathologist School of Medicine Tehran ...
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
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.