Title: Overview of Real Time PCR
1Overview of Real -Time PCR
- Real-Time PCR combines DNA amplification with
real time amplified product detection in a single
tube. - More specific then gel or hybridization assays.
- Less time consuming
- Quantitative results as opposed to
semi-quantitative or qualitative results. - Human (higher primate) or Y chromosome specific
- Monitors for PCR inhibition
2Introduction
- Methods based on changes in fluorescence
proportional to the increase in either specific
or non-specific PCR product. The fluorescence is
monitored during each PCR cycle to provide an
amplification plot allowing the user to follow
the reaction.
3Amplification Plot
4High Throughput Systems
- ABI Prism SDS 7000
- ABI Prism SDS 7700
- ABI 7300 and 7500 Real-Time PCR Systems
- iCycler iQ
- Mx 3000p Multiplex quantitative PCR system
5ABI Prism SDS 7000
- 96 well format
- Illuminated by a tungsten-halogen lamp
- 4 optical filters
- Optimized for FAM, SYBR green I, VIC, Yakima
Yellow, TAMARA and ROX dyes - Multiplexing capabilities
- Time 2 hours
- Display includes disassociation curve, and
results during the run
6ABI Prism SDS 7700/7900
- 96 well format.(7900 has 384 well format and
robotic capability). - Excitation is done by a laser.
- Fluorophores with an emission between 500 and 660
nm can be used. Includes FAM, TET, JOE, Yakima
Yellow, VIC, ROX, TAMARA, SYBR green I. - 2 hour run time.
7ABI 7300 and 7500 Real-Time PCR Systems
- 96 well plate format
- Excitation by Tungsten-Halogen lamp
- 25-100 µl reaction mix.
- 7300- FAM, SYBR green I VIC, Yakima Yellow, or
JOE, TAMARA, ROX - 7500- 5 color detection (NED, Cy3, Texas Red,
Cy5) better sensitivity with longer wavelengths - 2 hour run time
8iCycler iQ
- BioRad
- 96 well format
- PCR machine with an optical unit
- Halogen light source
- CCD camera measures emission from each well
simultaneously and detects 4 colors at once (FAM,
SYBR green I, VIC, Yakima Yellow, HEX, TET, Cy3,
Cy5, Texas Red, TAMARA, ROX - 2 hour run time
9Mx3000p Multiplex quantitative PCR system
- Stratagene
- Detects 4 fluorophores w/in the same reaction
using four different channels (customized
filters). Multiplexing ( 4 different targets at
the same time). - 96 well format
- Halogen lamp
- 350-750nm
- Detection- Scanning photomultiplier tube (PMT)
- Dyes- FAM, SYBR green I, TET, HEX, JOE, VIC,
Yakima Yellow, TAMARA, Cy3, Cy5, Texas Red, ROX,
Alexa Fluor 350 - Run Time 350
10Flexible Instruments
- LightCycler 2.0 Instrument
- Smartcycler
- RotorGene 3000 Four Channel Multiplexing System
- Used for multiplexing and performing multiple
experiments each with a different set of
parameters at the same time.
11Quantitative and Qualitative PCR Technologies
- Two types of Real-Time PCR detection chemistries
- 1. Specific Sequence Detection- Distinguishes
between a specific sequence of interest and
non-specific products. Can be used to detect
different alleles. - 2. Non-Specific Detection- Detects any dsDNA
produced during the reaction
12Non-Specific Detection Systems
- Uses a dsDNA intercalating dye that that
fluoresces once bound to DNA. The most common is
SYBR green 1. Excites at 497 nm and emits at 520
nm. - Do a melting curve start at 40C-95C. The
fluorescence will decrease when the dsDNA
disassociates. This depends on length and bp
composition. If the PCR reaction is optimized
will get a single peak corresponding to the
proper primer pair. Can be useful in quantifying
DNA. - Useful in optimizing a PCR reaction before
developing more expensive specific tests.
13Specific Detection Systems
- TaqMan Probes
- LNA Double Dye Oligonucleotide probes
- Molecular Beacon Probes
- Scorpions primers
14TaqMan Probes
- Double- Dye Oligonucleotides or dual labeled
probes. - Method of choice for forensic DNA quantitation
- Consists of a ssDNA probe that is complemenatry
to one of the amplicon strands - A fluorophore (FAM) is attached to one end of the
probe and a quencher (TAMARA) and a quencher to
the other end. The fluorophore is excited my the
instrument and passes its energy via FRET to the
quencher.
15FRET and NFQ
- Fluorescent Resonance Energy Transfer
- NFQ- Non- Fluorescent Quencher
16FRET Technology
- High energy fluorophore (reporter dye 6FAM) is
close to the NFQ and the energy will transfer
from high to low. As PCR products are formed the
specific TaqMAn probe will anneal to its
complementary sequence in the amplicon. Taq
Polymerase (due to its 5-3 nuclease activity)
will cleave the probe separating the fluorophore
(reporter) dye from the quencher. The
fluorescence is no longer quenched and is
proprotional to the number of amplicons produced.
17TaqMan Probe Mode of Action
18Uses for TaqMan Probes
- DNA Quantitation
- Mutation Detection-Probe designed to hybridize
over mutation - Allelic Discrimination
- Gene Expression
- Can be multiplexed
- Dark Quenchers- absorb emitted energy, but
release it as heat as opposed to fluorescence
19LNA Double Dye Oligonucleotide Probes
- LNA- Locked Nucleic Acids
- Integrating LNA bases into the DNA duplex of the
double dye oligonucleotide probes will change the
conformation from the B to the A form and
increase its stability, thus increasing its TM
value resulting in shorter, more specific probes. - Useful in applications requiring high affinity
probes such as SNP detection, gene expression
profiling and in situ hybridization.
20LNA Bases
- LNA is a bicyclic RNA analogue in which the
ribose moiety is structurally constrained by a
methylene bridge between the 2- oxygen and 4-
carbon atoms. When these LNA bases anneal with
the DNA bases they cause a conformational change
to occur in the helix.
21LNA Base Structure
22B and A Helices
23Molecular Beacon Probes
- Consist of a hairpin loop where the loop is a
single- stranded probe that is complementary to
the amplicon. The stem is usually 6 bases long
and consists mostly of C and Gs. It holds the
probe in the hairpin structure. A fluorphore is
attached to one of the stem and a quencher the
other. When the amplicon is produced during PCR,
the probe will bind to its specific target
sequence provided the probe-target duplex is
thermodynamically more stable then the hairpin
loop at the fluorescent acquisition temperature.
After the probe binds to its target the hairpin
is opened and the fluorophore and quencher are
separated.
24Continued
- The increase in fluorescence is reversible
because until the temperature is high enough the
hairpin loop will reform. This can produce a
melting curve that is useful for observing the
dynamics of the reaction and determining the best
temperature for fluorescence acquisition.
25Diagram of a Moleucular Beacon Probe
26Scorpion Primers
- PCR Primers with a stem-loop tail containing a
fluorophore and a quencher. The tail is separated
from the primer by a PCR blocker,a chemical
modification that prevents Taq from copying the
stem-loop tail of the Scorpion primer. This
prevents non-specific opening of the loop. The
PCR blocker links the loop to the 5 end of the
primer. The primer extends during the PCR
amplification and the specific probe sequence
will bind to its complement sequence. The hairpin
loop opens up during hybridization and the
fluorescence is no longer quenched. Because
enzymatic cleavage is not required the time it
takes to detect fluorescence is decreased. Main
advantage to TaqMan probes is low background and
fast reaction mechanism. Binding is more specific
then TaqMan probes and can produce a melt curve
analysis like Molecular Beacon probes. They can
be used in DNA quantitation, allelic
discrimination and mutation detection. Capable of
multiplexing.
27Scorpion Primer Reaction Diagram
28Technology Compatibility
29Fluorophore and Quencher
- The choice of which fluorophor and quencher
combination to use will give different results in
terms of sensitivity. - Fluorophore- a molecule that emits light of a
certain wavelength after having absorbed light of
a specific, but different wavelength first. The
emission wavelength is always higher than the
absorption wavelength. The fluorophore absorbs
light energy and is promoted to an excited state,
when it falls back to its ground state the excess
energy is released. - Quencher- a molecule that accepts energy from a
fluorophore in the form of light and disapates
this energy either in the form of light or heat.
30Fluorophore and Quencher Structures
31Fluorescence Principle
32Proximal and FRET Quenching
- Proximal- Fluorophore is in close proximity of
the quencher energy is transferred from
fluorophore to quencher and dissipates as heat
w/o fluorescence. Also called collisional
quenching. Dark Quenching. - FRET Quenching- The fluorophore transfers its
energy to the quencher (which can be another
fluorophore) the energy is released as
fluorescence of a higher wavelength. The
efficiency is related to the distance (more
precisely the Forster distance 1/r6, where r
the fluorophore distance) between the fluorophore
and quencher. TAMARA
33Proximal and FRET Quenching Diagram
34Optimal Fluorphore Quencher Combination
- The absorption spectrum of the quencher needs to
have good overlap with the emission spectrum of
the fluorphore to achieve optimal quenching.
Quenchers have a quenching capacity throughput
their absorption spectrum, but work best close to
their absorption maximum.
35Efficient Quenching of Fluorphores
- If the fluorescence is to high due to incomplete
quenching the relative fluorescence signal
(signal to noise ratio) will decrease. The
fluorophore-quencher combination which gives the
highest signal to noise ratio should be selected.
36ROX Passive Reference
- Used by ABI
- Correct for pipetting inaccuracies
- Does not participate in PCR
- Provides an internal fluorescent reporter to
which the reporter fluorescent can be normalized
37Rn and Bn
- Rn (normalized reporter signal) reporter
signal/ROX passive reference signal - This corrects for pipetting error only, not
background. - ?Rn is used to correct for background.
- ?Rn Rn Bn
- Bn (normalized baseline) baseline signal/ROX
passive reference - ?Rn (reporter signal/ROX passive reference
signal)- (baseline signal/ROX passive reference
signal - Thus the ROX passive reference signal influences
both the Rn and Bn
38Ct Value
- Ct value- The cycle in which a significant
increase in reporter signal above threshold can
be detected, i.e. the cycle in which the growth
curve crosses the threshold. - The automatic threshold is calculated by the
software and is defined as (average baseline
plus) (standard deviation of the baseline) - The ROX reference influences the baseline and
noise level. When the level of ROX increases, the
noise will decrease as will the ?Rn. It is
therefore important to determine the right level
of ROX that does not compromise ?Rn. - The Ct value is directly dependent on the
threshold level and indirectly dependent on
baseline level and ROX reference level. Minor
variations in ROX level can change Rn, Bn, ?Rn,
threshold and Ct. - Use a Master Mix that already contains ROX
39Quantitative PCR
- Quantitative PCR software uses the exponential
phase of PCR for quantitation. PCR is initially
an exponential process but reaches a plateau
phase as one or more of the reaction reagents
become limited. Reactions can plateau at
different levels even if they have the same
starting concentration of target. During the
exponential phase the amount of target is assumed
to be doubling every cycle w/o any bias from
limiting reagents. Analyses use the Ct value, the
point cycle number, at which the signal is
detected above the background and the
amplification is in the exponential phase. The
samples with the most abundant amount of DNA
template reach the Ct value earlier than the
samples with less starting DNA. The results are
normalized by comparing them against a standard
curve developed by running a series of known DNA
standards each of which has a different DNA
concentration.
40Caution Notes
- 1. DNA concentration results are dependent on
Molar Ratios and Molar Concentrations. When
comparing results, make sure that the reaction
volumes are the same. - 2. Be wary of comparing results from different
instruments or different platforms