PCR - POLYMERASE

CHAIN REACTION
Polymerase chain reaction : development of PCR
technique, steps of PCR: denaturation, annealing and
extension temperature, DNA polymerase, optimisation of
thermocycler, types of PCR, real time PCR, RT-PCR
 Introduction

• Invented by Kary Mullis in 1985.

• Kary Mullis, received the Nobel Prize in Chemistry in
1993.
• Without the ability to make copies of DNA molecules,
many forensic samples would be impossible to analyze.
 DNA from crime scenes is often limited in
both quantity and quality and obtaining a
cleaner, more concentrated sample is
normally out of the question (most
perpetrators of crimes are not surprisingly
unwilling to donate more evidence material to
aid in their prosecution).

 The PCR DNA amplification technology is

well suited to analysis of forensic DNA
samples because it is sensitive, rapid.
• PCR is an enzymatic process in which a specific region of
DNA is replicated over and over again to yield many copies
of a particular sequence.
• This molecular ‘Xeroxing’ process involves heating and
cooling samples in a precise thermal cycling pattern over
~30 cycles.
• During each cycle, a copy of the target DNA sequence is
generated for every molecule containing the target
sequence.
• The boundaries of the amplified product are defined by
oligonucleotide primers that are complementary to the 3'
ends of the sequence of interest.
• PCR is based on using the ability of DNA polymerase to
synthesize new strand of DNA complementary to the
offered template strand.
• Because DNA polymerase can add a nucleotide only
onto a preexisting 3'-OH group, it needs a primer to
which it can add the first nucleotide.
• This requirement makes it possible to delineate a specific
region of template sequence that the researcher wants to
amplify.
• At the end of the PCR reaction, the specific sequence will
be accumulated in billions of copies (amplicons).
• DNA Polymerases are a group of enzymes that catalyse
the synthesis of DNA during replication.
• The main function of DNA polymerases is to duplicate the
DNA content of a cell during cell division.
• They do so by adding nucleotides at 3’-OH group of the
growing DNA strand
 The strand that opens in the 3' to 5' direction towards the
replication fork is referred to as the lagging strand.
 The strand that runs in the 5' to 3' direction in the replication
fork is referred to as the leading strand.
PCR APPLICATIONS
• MOLECULAR BIOLOGY
• ENVIRONMENTAL SCIENCE
• FORENSIC/MEDICAL SCIENCE
• BIOTECHNOLOGY
• GENETICS
• GENE CLONING
• MICROBIOLOGY
The PCR Cycle

• Comprised of 3 steps:

• Denaturation of DNA at 950C

• hybridization ( annealing) at 40-500C
• DNA synthesis ( Primer extension) at 720C
• Denaturation (96°C): Heat the reaction strongly to
separate, or denature, the DNA strands. This provides
single-stranded template for the next step.
• Annealing (55 - 65°C): Cool the reaction so the primers
can bind to their complementary sequences on the single-
stranded template DNA.
• Extension (72°C): Raise the reaction temperatures so
Taq polymerase extends the primers, synthesizing new
strands of DNA.
• This cycle repeats 25- 35 times in a typical PCR reaction,
which generally takes 2 - 4 hours, depending on the
length of the DNA region being copied. If the reaction is
efficient (works well), the target region can go from just
one or a few copies to billions.
• That’s because it’s not just the original DNA that’s used as
a template each time. Instead, the new DNA that’s made
in one round can serve as a template in the next round of
DNA synthesis.
• There are many copies of the primers and many
molecules of Taq polymerase floating around in the
reaction, so the number of DNA molecules can roughly
double in each round of cycling.
• This pattern of exponential growth is shown in the image
below.
Components of PCR Reaction
• Template DNA
• Flanking Primers
• Thermo-stable polymerase
• Taq Polymerase
• dNTP Thermus aquaticus
• (dATP, dTTP, dCTP, dGTP)
The delicate balance of these 4 deoxyribonucleoside triphosphates
is important for the correct synthesis of DNA. adenine (dATP),
cytosine (dCTP), guanine (dGTP), and thymine (dTTP).
• PCR Buffer (mg++)
• Thermocyler
• PCR is a buffer that provides a suitable chemical
environment for activity of DNA polymerase.
• The buffer pH is usually between 8.0 and 9.5 and is often
stabilized by Tris-HCl.
• For Taq DNA polymerase, a common component in the
buffer is potassium ion (K+) from KCl, which promotes
primer annealing.
PCR

Primers, dNTPs, DNA, and

DNA polymerese added
PCR

Heated to 940C; DNA denatures

PCR

 Cooled to 600C, primers attach

 Raised to 720C, elongation
PCR

Thermal cycle repeated; multiple copies

Primers
• Paired flanking primers
• Length (17-28bp)
• GC content 50-60%
• Tm’s between 55-80
• Avoid simple sequences – e.g. strings of G’s
• Avoid primer self complementary
• e.g. hairpins, homodimers
PCR
Primer Specificity
• Universal – amplifies ALL bacterial DNA for instance
• Group Specific – amplify all denitrifiers for instance
• Specific – amplify just a given sequence
Forward and reverse primers
• Primer is an oligonucleotide sequence – will target a
specific sequence of opposite base pairing (A-T, G-C
only) of single-stranded nucleic acids
• If you know the sequence targeted for amplification, you
know the size which the primers should be annealing
across.
Basic rules for primer design
• Complementary to opposite strands with 3’ ends
pointing towards each other
• Be in vast excess
• unique
• no complementarity between 3’ ends (primer
dimers)
• perfect match with 3’ end (additions to 5’ end)
PCR

Primer design

Primer Size: Too small, may bind to more

than one site in the genome

Too Large? Yes. Long primers take a

longer time to hybridize and would slow
down the PCR cycle. (not > 30 bp)
PCR

Primer design
 Not all primers will work the same; primers
perform differently at different temperatures
 Annealing temperature is most important.
 Too low = non-specific binding
 Too high = primer will not bind

 Ideal annealing temperature can be

mathematically estimated
PCR

Primer design (temperature calculation)

 Melting temperature (Tm) is temperature
at which the primer will dissociate.
 Annealing temperature should be just
1-2oC below Tm.
 39

Annealing Temperature
- Very important since the success and
specificity of PCR depend on it because
DNA-DNA hybridization is a temperature
dependent process.
- If annealing temperature is too high,
pairing between primer and template DNA
will not take place then PCR will fail.
- Ideal Annealing temperature must be low
enough to enable hybridization between
primer and template but high enough to
prevent amplification of nontarget sites.
- Should be usually 1-2° C or 5° C lower than
melting temperature of the template-
 DNA Polymerase
• Most proteins denature at extreme pH or high temperatures.
• DNA Polymerase is the enzyme responsible for copying the
sequence starting at the primer from the single DNA strand
• Human DNA polymerase would denature at 94C. New
polymerase would have to be added at each elongation step.
• Commonly use Taq, Taq polymerase is the DNA polymerase I
an enzyme from the hyperthermophilic organisms Thermus
aquaticus, isolated first at a thermal spring in Yellowstone
National Park
• Many of its enzymes will not denature at high temperatures.
• This enzyme is heat-tolerant  useful both because it is
thermally tolerant (survives the melting T of DNA
denaturation) which also means the process is more specific,
higher temps result in less mismatch – more specific
replication
Heat-stable polymerase is vital to the
ease of the process…
PCR Polymerases
• Taq(From Thermus aquaticus), Vent(From
Pyrococcus furiosus), Pfu (From Thermococcus
litoralis), others
• Native or Cloned
 PCR Buffer

• Basic Components
• 20mM Tris-HCL pH 8.4
• 50mM KCl
• 1.5 mM MgCl2

• Magnesium –
• Since Mg ions form complexes with dNTPs, primers and DNA
templates, the optimal concentration of MgCl2 has to be selected
for each experiment.
Too few Mg2+ ions result in a low yield of PCR product,
and too many increase the yield of non-specific products and
promote mis-incorporation.
• Typical Reaction

• 32.5 μl dH2O
• 5 μl 10 X PCR buffer + mg
• 1 μl 200 μM dNTP
• 0.5 μl 50 μM Left Primer
• 0.5 μl 50 μM Right Primer
• 10 μl Worm or Fly Lysate
• 0.5 μl Taq Pol (5 Units/ μl)
50 μl Total Vol
 Master Mix
• 1 volume master mix
• 32.5 μl dH2O
• 5 μl 10 X PCR buffer
• 1 μl 200 μM dNTP
• 0.5 μl Taq Pol (5 Units/ μl)
39 μl Total Volume
• To set up 4 reactions prepare 4.4 volumes of reaction master mix
– 143 μl dH2O
– 22 μl 10 X PCR buffer
– 4.4 μl 200 μM dNTP
– 2.2 μl Taq Pol (5 Units/ μl)
• Individual reactions
- 39 μl master mix
- 0.5 μl Left primer
- 0.5 μl Right primer
- 10 μl worm lysate
 Templates for PCR
• Dried blood
• Semen stains
• Vaginal swabs
• Single hair
• Fingernail scrapings
• Insects in Amber
• Egyptian mummies
• Buccal Swab
• Toothbrushes
Contamination!
• strategies for eliminating contamination (or reducing
effects)
• physical separation of product analysis from reaction set up
• controls
PCR Variables
1. Temperature
2. Cycle Times and Temps
3. Primer
4. Buffer
5. Polymerase
 Temperature

• Denaturation
• Trade off between denaturing DNA and
not denaturing Taq Polymerase
• Taq half-life 40min at 95 °, 10min at
97.5°
• 95°
• Annealing
• Trade off between efficient annealing
and specificity
• 2-5 ° below Tm
• Extension
• Temperature optimum for Taq
Polymerase
• 72 °
Cycle Times and Temps

Typical PCR Run

Step Time/Temp
1 3 min at 95°
2 30 sec at 95°
3 1 min at 55°
4 2 min at 72°
5 Go to step 2 - 29 times
6 8 min 72°
7 0 min 4°
8 End
PCR
PCR

Studying PCR products

1. Gel Electrophorsis (slab or capillary)

2. Cloning of PCR products
3. Sequencing of PCR products
PCR

Gel Electrophoresis

 Success of PCR
reaction determined by
running an agarose gel.
 PCR products
separated by length
PCR

1. Correct size
2. Absent = reaction didn’t work
3. Wrong size = wrong region amplified
4. Multiple bands = non-specific primer binding
PCR

Gel Electrophoresis

If screening colonies for presence of

gene, sometimes presence/absence
of band is the extent of the test.
APPLICATIONS OF PCR
PCR can be applied in many fields
• In diagnosis therapy
• In nucleic acid detection assays
• In medical field
• In agricultural sciences
• In mycology-parasitology
• In dentistry
• In virological diagnostics
• In insert analysis
• In molecular systematic evolution
• In cancer therapy
• In therapy resistant assessment
• PCR-as biomarker
• In forensic medicine
• In virology
• In bacteriology
• In phytopathology
• In PCR-fingerprinting
• In the detection of microbiological gene.
 DIAGNOSTIC APPLICATIONS: Quantitative HIV viral load
determination is a most important parameter for diseases
outcome and used for diagnosis of the diseases.
 In Medicine: culturing and identification are reduced by the
usage of PCR technique.
 In Forensic Science: Genetic basis of diseases with
sudden death can be investigated with molecular methods.
 Genetic profile of alleles identified in different regions of
DNA marked by genetic marker STR( short Tandem
Repeat).
In Plant Research: In plant research it can be used in
Insert analysis, Phyto-pathology
 Molecular system and evolution
Types of PCR
• Real-time PCR
• Quantitative real time PCR (Q-RT PCR)
• Reverse Transcriptase PCR (RT-PCR)
• Multiplex PCR
• Nested PCR
• Long-range PCR
• Single-cell PCR
• Fast-cycling PCR
• Methylation-specific PCR (MSP)
• Hot start PCR
• High-fidelity PCR
• In situ PCR
• Variable Number of Tandem Repeats (VNTR) PCR
• Asymmetric PCR
• Repetitive sequence-based PCR
• Overlap extension PCR
• Assemble PCR
• Intersequence-specific PCR(ISSR)
• Ligation-mediated PCR
• Methylation –specific PCR
• Mini primer PCR
• Solid phase PCR
• Touch down PCR
• Quantitative PCR techniques It is also referred to real
time PCR.
• Qualitative PCR techniques When PCR techniques is
used for detecting a specific DNA segment, it is called as
quantitative PCR method.
• Conventional PCR:the primers bind specifically to each
other with 2 DNA strands.
• Multiplex Pcr technique detects different pathogens in a
single sample, used to identify exonic/intronic sequence 5
in specific genes.
• Nested PCR is used because it intends to reduce the
contaminations in products due to the amplification of
unexpected primer binding sites.
 Real time PCR
• A real-time polymerase chain reaction (real-time PCR)
(Sometimes referred to as qPCR) is laboratory
technique of molecular biology based on the polymerase
chain reaction (PCR).
• It monitors the amplification of a targeted DNA molecule
during the PCR (i.e., in real time), not at its end, as in
conventional PCR.
• Amplification is the prime goal of any PCR reaction.
• Using dNTPs, primers and PCR reaction buffer, the Taq
DNA polymerase amplifies our DNA in vitro.
• The same process when occurs in vivo, known as
replication.
• In the traditional PCR method after the amplification, the
PCR products or the amplicon are run on the agarose gel
or PAGE to detect the presence or absence of DNA
amplification. But in the real-time PCR monitor
amplification after each PCR cycle in a real-time manner.
• A camera or detector detects each amplicon produced
during the amplification by measuring the fluorescence
emitted.
• Usually, the chemistry behind real-time monitoring relies
on the use of fluorescent dye.
• As the amplification progresses, the detector detects the
amount of fluorescence emitted.
• Two common methods for the detection of PCR products
in real-time PCR are
• (1) non-specific fluorescent dyes that intercalate with any
double-stranded DNA and
• (2) 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.
• Real-time PCR is carried out in a thermal cycler with the
capacity to illuminate each sample with a beam of light of at
least one specified wavelength and detect the fluorescence
emitted by the excited fluorophore.
• The thermal cycler is also able to rapidly heat and chill
samples, thereby taking advantage of the physicochemical
properties of the nucleic acids and DNA polymerase.
Principle of real-time PCR
• “The amount of the nucleic acid present into the sample is
quantified using the fluorescent dye or using the
fluorescent-labeled oligos.”
• When a dye or probe binds with the target template, it
releases a fluorochrome which resultantly emits
fluorescence for the detector to detect.
• The detector captures a signal as a positive template
amplification.
 69

 Quantitative real time PCR (Q-RT PCR)

It is used to amplify and also for quantification and detection

of DNA sample.
Real time PCR using DNA dyes
Fluorescent reporter probe method
-Detection and quantitation of fluorescent reporter the
signal of which increases in direct proportion to the
amount of PCR product in a reaction
-Does not measure the amount of end product but its
production in real time
 Labelling approaches
SCYBR
Y B R G r eengreen
I
Real-Time PCR
hn
hn

 This technique allows quantitation of

DNA and RNA. dsD N A -- bound dye > 100
ssD N A -- unbound dye
 Reactions are characterized by the m inim a l fluoresce nce fold inc rea se fluore scenc e

point in time during cycling when TAQ-man probes

T aq M an -- H yd rolysis P rob e

amplification of a PCR product is

hn
hn
first detected rather than the amount fluor quenche r

of PCR product accumulated after a

fixed number of cycles. E xte nsion continues

 The higher the starting copy number

FRET probes
H yb rid iz ation p rob es
of the nucleic acid target, the sooner
FRET
a significant increase in hn
hn

fluorescence is observed.
donor acc eptor
M onitor acce ptor fluorescence

Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe

72
Steps of Real time- PCR
• A. Amplification
• B. Detection
• The detection is based on fluorescence technology.
• The specimen is first kept in proper well and subjected to
thermal cycle like in the normal PCR.
• Real Time PCR is subjected to tungsten or halogen
source that lead to fluoresce the marker added to the
sample and the signal is amplified with the amplification of
copy number of sample DNA.
• The emitted signal is detected by an detector and sent to
computer after conversion into digital signal that is
displayed on screen.
• The signal can be detected when it comes up the
threshold level (lowest detection level of the detector).
• It is a technique used to monitor the progress of a PCR
reaction in real-time. At the same time, a relatively small
amount of PCR product (DNA, cDNA or RNA) can be
quantified. It is based on the detection of the fluorescence
produced by a reporter molecule which increases, as the
reaction proceeds.
• It is also known as a quantitative polymerase chain
reaction (qPCR), which is a laboratory technique of
molecular biology based on the polymerase chain
reaction (PCR).
• qPCR is a powerful technique that allows exponential
amplification of DNA sequences.
• A PCR reaction needs a pair of primers that are
complementary to the sequence of interest.
• Primers are extended by the DNA polymerase.
• The copies produced after the extension, so-called
amplicons, are re-amplified with the same primers leading
thus to exponential amplification of the DNA molecules.
• After amplification, however, gel electrophoresis is used to
analyze the amplified PCR products and this makes
conventional PCR time consuming; since the reaction
must finish.
RT-PCR
• transcription polymerase chain reaction (RT-PCR) is a
laboratory technique combining reverse
transcription of RNA into DNA (in this context
called complementary DNA or cDNA) and amplification of
specific DNA targets using polymerase chain
reaction (PCR).
• It is primarily used to measure the amount of a specific
RNA.
• In RT-PCR, the RNA template is first converted into
a complementary DNA (cDNA) using a reverse
transcriptase (RT).
• The cDNA is then used as a template for exponential
amplification using PCR.
• This is achieved by monitoring the amplification reaction
using fluorescence, a technique called real-time PCR or
quantitative PCR (qPCR).

• Combined RT-PCR and qPCR are routinely used for

analysis of gene expression and quantification of viral
RNA in research and clinical settings.
 81

 REVERSE TRANSCRIPTASE PCR

-It
is employed for
amplification of RNA
molecules .

-RT-PCR is widely used

in expression profiling, to
determine the expression
of a gene or to identify the
sequence of an RNA
transcript.
RT-PCR
The enzyme reverse transcriptase is used to make a DNA copy (cDNA)
of an RNA template from a virus or from mRNA.
Viral RNA Bacterial mRNA Protozoan (eukaryotic) poly A mRNA

3’
AAAA

Reverse transcriptase
RNA Primer
5’
5’
3’
Extension

3
’
5
’ RNA Normal PCR with two primers

3
’ cDN
A
5
’
 83

VARIATIONS OF PCR
PCR is highly versatile technique and has been
modified in variety of way to suit specific
applications.

Inverse PCR
-In this method amplification of DNA of unknown
sequence is carried out from known sequence.
- This is especially useful in identifying flanking
sequences of various genomic inserts.
Multiplex PCR

Use of multiple sets of primers to detect more than one organism or to

detect multiple genes in one organism.
Remember, the PCR reaction is inherently biased depending on the G+C
content of the target and primer DNA. So performing multiplex PCR can
be tricky.

E. Coli Salmonella sp.

genome genome

or
 85

-TaqMan probes are designed such that they anneal within a DNA
region amplified by a specific set of primers.

-As the Taq polymerase extends the prime rand synthesizes the
nascent strand, the 5' to 3‘ exonulease activity of the polymerase
degrades the probe that has annealed to the template.

- Degradation of the probe releases the fluorophore from it and

breaks the close proximity to the quencher, thus relieving the
quenching effect and allowing fluorescence of the fluorophore.

-Fluorescence detected in the real-time PCR thermal is directly

proportional to the fluorophore released and the amount of
DNA template present in the PCR.
86
 ASYMMETRIC PCR 87

-It is used for synthesis of Single stranded DNA molecules

useful for DNA sequencing
-The two primers are used in the 100:1 ratio so that after 20-25
cycles of amplification one primer is exhausted thus single
stranded DNA is produced in the next 5-10 cycles.

 Allele- Specific PCR

-Selective PCR amplification of the alleles to detect single
nucleotide polymorphism (SNP)
-Selective amplification is usually achieved by designing a
primer such that the primer will match or mismatch one of
the alleles at the 3’ end of the primer.
• In a real time PCR assay a positive reaction is detected by
accumulation of a fluorescent signal.
• The Ct (cycle threshold) is defined as the number of cycles
required for the fluorescent signal to cross the threshold (ie
exceeds background level).
• Ct levels are inversely proportional to the amount of target
nucleic acid in the sample (ie the lower the Ct level the greater
the amount of target nucleic acid in the sample).
• Cts < 29 are strong positive reactions indicative of
abundant target nucleic acid in the sample
•
Cts of 30-37 are positive reactions indicative of moderate
amounts of target nucleic acid
•
Cts of 38-40 are weak reactions indicative of minimal
amounts of target nucleic acid which could represent an
infection state or environmental contamination.
PCR qPCR
It is an advanced PCR method which is
PCR is used to analyse a short stretch of
used to amplify as well as quantify the
DNA by amplification
amount of DNA
In qPCR, primer, as well as fluorescent
n PCR, primer is used for polymerisation
probes or dyes, are used
Results are investigated by gel Fluorescence emitted by dye or probe is
lectrophoresis recorded during the PCR process
The data is recorded during the
The data is recorded at the end of the
amplification process at the exponential
rocess
phase
Ethidium bromide is used to stain the DNA Fluorescent dyes or DNA probes labelled
ragments with a fluorescent reporter are used
is a low-resolution technique It is a high-resolution technique
Distinct bands of various DNA fragments Different peaks related to different DNA
an be seen on agarose gel fragments are seen during qPCR
Conventional PCR takes more time to
qPCR takes less time to deliver the result
enerate a result
It is used to quantify the DNA present in the
is used to detect the presence or absence
sample. It is used to analyse gene
f DNA in the sample, detect gene
What is RT-PCR?

• RT-PCR or reverse transcription PCR comprises two

steps, the first is the reverse transcription process, and
the second is the amplification of the desired DNA
sequence by polymerase chain reaction or PCR
• . RT-PCR is used to detect the RNA in a sample.
• The amount of RNA can be measured by using RT-
qPCR.
• RT-PCR combined with qPCR (RT-qPCR) is very useful in
doing quantitative analysis of viral RNA and gene
expression.
• The steps of RT-PCR are the same as PCR, with the
additional first step of reverse transcription. In RT-PCR,
complementary DNA (cDNA) is produced first using
reverse transcriptase (RT).
• The cDNA, thus formed, is then used as a template for the
standard amplification process by PCR.
• To sum up, RT-PCR and qPCR are the advanced
methods of PCR or polymerase chain reaction.
• qPCR gives faster, more detailed real-time results and is
used to quantify nucleic acids. RT-PCR is used to detect
and amplify cDNA.
• qPCR or quantitative PCR is also referred to as real-time
PCR.
• It gives an additional quantitative analysis of the DNA or
RNA in RT-qPCR.
• In qPCR or real-time PCR, as the name suggests, the
amplification can be monitored as the PCR progresses.
• In qPCR, fluorescent labelling allows real-time data
collection as a polymerase chain reaction is performed.
• PCR products can be detected in real-time in qPCR by two
methods:
• Using non-specific fluorescent dyes helps in detecting double-
stranded DNA. The dsDNA binding dye gives fluorescence
signals as the DNA is amplified, and it increases after each
cycle.
• The disadvantage of this method is that only one target can be
examined at a time as it binds to all dsDNA fragments.
• Using fluorescent-labelled DNA probes, which are sequence-
specific.
• They can be detected after hybridization with its
complementary sequence.
• As the DNA probes are target specific, many targets can be
analysed simultaneously
• 1) Primer design
• Design and synthesize the primers of the target gene.
• Primers around 20~25 nucleotides in length should be
synthesized.
• The GC content of these primers is designed as similar as
possible.
• (2) RNA extraction
• Total RNA is extracted from cells or tissues
of the plants or animals taking a standard extraction
protocol with Trizol, dissolved in DEPC-treated deionized
water and quantified with spectrophotometer.
• 3) Reverse transcription(RNA→cDNA)
• RT-PCR is used to clone expressed genes by reverse
transcribing the RNA of interest into its DNA complement
through the use of reverse transcriptase.
• Subsequently, the newly synthesized cDNA is amplified
using traditional PCR. The total RNA is used as a
template and Oligo (dT) or random primers and reverse
transcriptase are used to reverse transcription into cDNA.
• (4) Real-time PCR
• Real-time PCR (qPCR) is the method of choice for
quantification of gene expression.
• It is the preferred method of obtaining results from array
analysis and gene expressions on a global scale.
• Currently, there are four different fluorescent DNA probes
available for the real-time PCR products detection: SYBR
Green, TaqMan, Molecular Beacons, and Scorpions.
• While the SYBR Green dye emits its fluorescent signal
simply by binding to the double-stranded DNA in solution,
the TaqMan probes, Molecular Beacons and Scorpions
generation of fluorescence depend on Förster Resonance
Energy Transfer (FRET) coupling of the dye molecule and
a quencher moiety to the oligonucleotide substrates.
• (5) Result analysis
• In the initial few cycles of the qPCR amplification reaction,
the fluorescence signal changes little and approaches a
straight line, which is the baseline.
• The fluorescence signals of the original 15 cycles is the
background signal of fluorescence.
• The fluorescence domain values are 10 times the
standard deviation of 3-15 circular fluorescent signal, and
threshold set in the exponential phase of the PCR
amplification.
• The CT value indicates the number of cycles experienced
by each PCR reaction tube when the fluorescence signal
reaches the set domain.
• The more number the template onset copy has, and less t
he Ct value will be and vice versa. Generating consistent
amplification across a wide dynamic range is fundamental
to qPCR methodology, and use the △△t method for gene
expression analysis.