Advanced PCR: Methods and Applications: Dr. Maryke Appel
Advanced PCR: Methods and Applications: Dr. Maryke Appel
Advanced PCR: Methods and Applications: Dr. Maryke Appel
A quick review of “standard” PCR
An enzyme for every need
The third pillar: RTases & RT-PCR
The next generation: Real-time PCR
The sky is (not) the limit
What we do for a living
Standard PCR
PCR is a cornerstone of modern molecular biology
The relatively simple concept of DNA amplification: cycles of denaturation, primer annealing, and extension, is
being utilized in a myriad of applications:
Diagnostics (nucleic acid testing, pathogen detection)
Advanced research (gene expression)
Genomics (sequencing, genotyping, whole genome amplification)
EXPONENTIAL
AMPLIFICATION
Standard PCR
These enzymes are not thermostable, have low replicating fidelity, and possess
RNase activity.
Conversion of mRNA to cDNA by Reverse Transcription
RT-PCR CONSIDERATIONS:
The quality and purity of the starting RNA template is crucial to the success of RT-PCR.
Total RNA or poly(A)+ RNA can be used as the starting template - both must be intact and free
of contaminating genomic DNA.
Specific capture of poly(A)+ RNA will enrich a targeted message so that less of the reverse
transcription reaction is needed for the subsequent amplification.
The efficiency of the first-strand synthesis reaction, which can be related to the quality of the
RNA template, will also significantly impact the results of the subsequent amplification.
Real-Time PCR
QUANTITATION
Real-Time PCR
PCR was traditionally limited to end-point analysis using
agarose gels
Limitations of end-point PCR:
Poor precision
Low sensitivity
Short dynamic range
Low resolution
Size-based discrimination
Ethidium bromide for staining does not allow for
accurate quantitation
Requires post-PCR processing
Real-Time PCR
Plateau
phase
End Point
Linear
phase
Exponential
phase
Cycle threshold is
related to the initial
target copy number
CT
Real-Time PCR
Advantages of real-time vs. end-point PCR:
•
Collects data in the exponential growth phase (vs end-point plateau)
•
Increase in fluorescent signal is proportional to number of amplicons generated
•
Increased dynamic range of detection
•
Does not require post-PCR processing
•
Increased sensitivity (detection down to 2-fold change)
Applications:
•
Viral quantitation
•
Quantitation of gene expression
•
Microarray verification
•
Drug therapy efficacy
•
Pathogen detection
•
Genotyping
Real-Time PCR
Detection Assays: SYBR Green Dye
SYBR Green I binds to double-
stranded DNA. The resulting DNA-
dye-complex absorbs blue light
(max = 498 nm) and emits green
light
(max = 522 nm)
As DNA is amplified SYBR
fluorescence increases proportionally
Non-specific dye used to detect the
presence or absence of an amplicon
Non-target sequence-specific detections
systems are susceptible to false-
positives
Real-Time PCR
Detection Assays: Sequence-specific probes
5’3’ exonuclease activity of
DNA polymerase cleaves
reporter
dye from quencher and
allowing
fluorescence.
Specific sequences are able to
beTaqMan improves:
detected in the real-time
Specificity
reaction.
Product quantification
Multiplex PCR
Advanced PCR
PCR has become a central tool for DNA analysis across all disciplines of
biology and biochemistry
Novel enzymes and instrumentation are creating new applications for
PCR
Other advanced PCR methods for research and diagnostic applications:
Hot start PCR (specificity)
Cycling sequencing (DNA sequencing)
Site-directed mutagenesis PCR
Colony PCR
Multiplex-PCR
Error-prone PCR (mutagenesis)
StEP PCR (recombination)
Emulsion PCR (cell-free cloning)
What do we do at Kapa Biosystems?
We are developing a suite of “standard” enzymes for PCR:
Taq DNA polymerase
Type B polymerases (Hi-Fi): Pfu, KOD, chimera
Hot-start Taq and Type B polymerases
Long-range PCR blends