Various Platforms of PCR

Various platforms of PCR

1) Allele-specific PCR
2) Real-time PCR
3) Digital PCR
4) Hot start PCR
5) In silico PCR
6) Multiplex PCR
7) Nested PCR
 Allele-specific PCR
• Allele-specific PCR concept:
• Primer that forms a 3′ mismatch with the DNA
template can be refractory to primer
extension by DNA polymerase.
• Refractory; hard to work.
• Oligonucleotide primers specific for all known
alleles can be synthesized and used to detect
the alleles in DNAs of unknown genotype.
3’ mismatch
Wild type assay mix 1) dH2O Mutant type assay mix
2) DNA template
3) Forward wild type primer
4) Reverse common primer
5) dNTP
6) MgCl2
7) Taq polymerase

1) dH2O
2) DNA template
3) Forward mutant type
primer
4) Reverse common primer
5) dNTP
6) MgCl2
7) Taq polymerase
 Gene: Human ABCA7
SNP: rs3764650

GCGAACTTTGCACC[G/T]TTACACCACTCCAC

T T

Homozygous wild type (TT)

ABCA7 rs3764650 Heterozygous (TG)

T G

Homozygous variant (GG)

G G
Wild-type Homozygous wild-type Variant-type
assay mix (TT) assay mix

T T T T

Chromosome 19 Chromosome 19

T T T T
A A A A
T T G G

369 bp (√) 369 bp (√) 369 bp (X) 369 bp (X)

846 bp (√) 846 bp (√) 846 bp (√) 846 bp (√)

The expected PCR bands

Validation: DNA sequencing
WT lane VT lane
846 bp

369 bp
Wild-type Heterozygous Variant-type
assay mix (TG) assay mix

T G T G

Chromosome 19 Chromosome 19

T G T G
A C A C
T T G G

369 bp (√) 369 bp (X) 369 bp (X) 369 bp (√)

846 bp (√) 846 bp (√) 846 bp (√) 846 bp (√)

The expected PCR bands

Validation: DNA sequencing
WT lane VT lane
846 bp

369 bp
Wild-type Homozygous variant-type Variant-type
assay mix (GG) assay mix

G G G G

Chromosome 19 Chromosome 19

G G G G
C C C C
T T G G

369 bp (X) 369 bp (X) 369 bp (√) 369 bp (√)

846 bp (√) 846 bp (√) 846 bp (√) 846 bp (√)

The expected PCR bands

Validation: DNA sequencing
WT lane VT lane
846 bp

369 bp
 Real-time PCR
• Real-time PCR is used to amplify and
simultaneously detect or quantify a
targeted DNA molecule.
• Real-time PCR follows general principle of PCR.
Additional feature is the amplified DNA is
detected as the reaction progresses in "real
time“.
• Real-time PCR is quantitative PCR.
• Compared to standard PCR, where the product of
the reaction is detected at its end.
 Real-time PCR
• Two common methods for the detection of
products in quantitative PCR are:
– non-specific fluorescent dyes that intercalate with
any double-stranded DNA (Eg. SYBR green).
– sequence-specific DNA probes consisting of
oligonucleotides that are labelled with a
fluorescent reporter which permits detection only
after hybridization of the probe with its
complementary sequence to quantify messenger
RNA (mRNA) (Eg. TaqMan).
SYBR Green

4
SYBR Green

7
TaqMan

3
TaqMan
TaqMan

6
TaqMan

8
Conventional PCR Real-time PCR
machine machine
Analysis in real-time PCR machine
using computer
 Digital PCR
• Digital PCR is used to quantify and
clonally amplify nucleic acids
including DNA, cDNA or RNA. A
sample is partitioned into thousands
of separate reaction chambers.
• So that individual nucleic acid
molecules within the sample are
localized and concentrated within
many separate regions.
• The partitioning of the sample allows
one to estimate the number of
different molecules by assuming that
the molecule population.
 Digital PCR
• The partitions are cycled with a
conventional thermal cycling protocol,
and taqman hydrolysis probes are the
method by which specific
fluorescence is detected.
• After PCR amplification, nucleic acids
are quantified by counting the regions
that contain PCR end-product, positive
reactions.
• Poisson statistics are used to estimate
DNA, cDNA or RNA concentration.
dPCR
Digital PCR
Digital PCR
 dPCR vs qPCR
qPCR
• Real-time PCR is a widely used method for quantifying DNA or RNA in a sample.
• It monitors the amplification of the target molecule in real-time during the PCR reaction using
fluorescent probes or dyes.
• These probes emit fluorescence as they bind to the target molecule, and the fluorescence signal is
measured at each cycle of amplification.
• The fluorescence intensity is directly proportional to the amount of target molecule present in the
sample, allowing for real-time monitoring and quantification.

dPCR
• Digital PCR, on the other hand, does not rely on measuring fluorescence intensity to quantify the
target molecule.
• Instead, it partitions the sample into numerous individual reactions (often referred to as droplets or
chambers), and each partition is then analyzed separately to determine whether or not it contains
the target molecule.
• The partitions are typically read using fluorescence or other methods to detect the presence or
absence of the target molecule. The number of positive partitions is then used to calculate the
absolute quantity of the target in the original sample.

Conclusion
• Real-time PCR provides quantitative results based on fluorescence intensity, whereas digital PCR
provides absolute quantification by analyzing the presence or absence of the target molecule in
individual partitions.
 Hot start PCR
• In conventional PCR, the Taq DNA polymerase is
active at room temperature and to a lesser
degree, even on ice. In some instances, when all
the reaction components are put together,
nonspecific primer annealing can occur due to
these low temperatures.
• This nonspecific annealed primer can then be
extended by the Taq DNA polymerase, generating
nonspecific products and lowering product yields.
 Hot start PCR
• Hot start PCR significantly reduces nonspecific
priming, the formation of primer dimers, and
often, increases product yields.
• Specific antibodies are used to block taq-
polymerase at annealing temperature.
• So, when the temperature raises for
amplification to 72 , the specific antibody
detaches from taq-polymerase and the
amplification with greater specificity starts.
Hot start PCR
 In silico PCR
• What is in silico?
• Performed on computer, through computer
simulation.
• In silico PCR refers to computational tools
used to calculate theoretical PCR results using
a given set of primers to amplify DNA
sequences from a sequenced genome or
transcriptome.
 Multiplex PCR
• Different between singleplex and multiplex PCR
– Singleplex PCR refers to amplifying one DNA template with
one set of primers in one reaction.
– Multiplex, amplifying DNA templates with more than one
sets of primers in one reaction.
• Multiplex PCR consists of multiple primer sets within a single
PCR mixture to produce amplicons of varying sizes that are
specific to different DNA sequences.
• By targeting multiple genes at once, additional information
may be gained from a single test run that otherwise would
require several times the reagents and more time to perform.
 Multiplex PCR
• Annealing temperatures for each of the primer
sets must be optimized to work correctly within a
single reaction.
• In terms of amplicon sizes, their base pair length,
should be different enough to form distinct bands
when visualized by gel electrophoresis.
• Commercial multiplexing kits for PCR are
available and used by many forensic laboratories
to amplify degraded DNA samples.
singleplex and multiplex PCR
 Nested PCR

• A commonly occurring
problem in PCR is primers
binding to incorrect regions
of the DNA, giving
unexpected products.
• Nested PCR functions to
reduce non-specific binding
in products due to the
amplification of unexpected
primer binding sites.
• Normally two sets of primer
are required in nested PCR.
• The second set intended to
amplify a secondary target
within the first run product.