Advanced PCR:

Methods and Applications

Dr. Maryke Appel

Senior Scientist, Kapa Biosystems
 Overview


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

PCR remains a complex reaction involving ionic interactions, kinetic

constants, and enzymatic activities.
In addition to primer design, many applications still require the
optimization of buffer conditions, type of enzyme, and cycling
parameters.

Buffer considerations: Template DNA, primers

•
Mg2+ concentration Reaction buffer
•
KCl Enzyme
•
Ammonium sulfate
•
pH
DNA polymerases : Standard PCR
Polymerase enzymes are responsible for DNA replication in the reaction

Standard PCR : Taq polymerase

Taq is a Type A polymerase isolated from the eubacterium
Thermus aquaticus capable DNA polymerization in the 5’3’
direction.
o
Thermostable
o
Generally robust, requires minimal optimization
o
High yield of DNA targets <5 kb
o
Low fidelity (5 x 10-5)
o
Difficult to use with complex template, secondary
structures (GC-rich, homopolymeric regions)
o
Sensitive to inhibitors such as salt, blood, phenol,
fluorescent dyes
DNA polymerases : Hi-Fidelity PCR
Polymerase enzymes are responsible for DNA replication in the reaction

High Fidelity PCR : Pfu polymerase

Pfu is a Type B polymerase isolated from archaeal
bacteria Pyrococcus furiosus, capable DNA polymerization
in the 5’3’ direction as well as a 3’5’ exonuclease
activity (proofreading).
o
Highly thermostable
o
3’5’ nuclease activity removes mismatches and
greatly improves the fidelity of the reaction (2.2 x 10 -6)
o
Used when the amplification product will be cloned
o
Requires optimization, less robust
o
Typically lower yields
 DNA polymerases : Long-range
PCR
During replication Taq will occasionally incorporate a base mismatch and not be
able to extend. The probability of this happening increases with target length

Standard PCR High Fidelity PCR

Taq polymerase Pfu polymerase

Long Range PCR

Blend of Taq + small amount of Pfu

Allows for amplification of targets >20 kb

 The 3rd pillar: Reverse
transcriptase
 The thermostable DNA polymerases used in standard PCR require a DNA template and is
therefore limited to the analysis of DNA samples.
 The analysis of differential expression of genes and the cloning of cDNAs from rare messages
requires RNA template.
 In order to apply PCR to the study of RNA, the RNA sample must first be reverse transcribed
to cDNA using a reverse transcriptase enzyme (RNA-dependent polymerase):

- Avian myeloblastosis virus (AMV)

- Moloney murine leukemia virus (M-MLV)

These enzymes are not thermostable, have low replicating fidelity, and possess
RNase activity.
Conversion of mRNA to cDNA by Reverse Transcription

or: gene-specific primer

or: random hexamers
 RT-PCR

RNA cDNA DNA for analysis

Reverse Transcription PCR

RTase DNA polymerase

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 instruments and chemistry

allow for the detection and quantitation
of amplification throughout the reaction
 Standard PCR

Real-Time PCR
Plateau
phase
End Point
Linear
phase

Exponential
phase

Area of detection for

real-time PCR
 Real-Time PCR
QUANTITATION

Theoretically there is a quantitative relationship between the amount of starting sample and the
amount of PCR product at any given sample.

Real-time PCR detects the accumulation of amplicon during the reaction. The data is then measured
at the exponential phase of the PCR reaction rather than end-point plateau. The exponential phase
is the optimal point for analyzing data.

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

Dual fluorophore-labeled oligonucleotide

probe: e.g. TaqMan


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

Really focuses on the engineering of novel polymerases for advanced

applications:
 2nd generation Taq
 SYBRTaq
 Ultra-high fidelity Type B