Welcome back to Applied Biological Materials PCR video series! In this video, we will introduce
the two major types of qPCR, the SYBR Green base qPCR, and the Taqman base qPCR. For all
your PCR needs, at abm, or Applied Biological Materials, we offer a wide range of DNA polymerases, PCR
Mastermixes, and other PCR products. qPCR, also known as real-time PCR or quantitative PCR is
a technique that is able to amplify and detect changes in the amplicon concentration. qPCR
collects data during amplification by using fluorescence signal emitted by special probes
or DNA binding dyes. Over the years, qPCR has been utilized for many applications. It
is mostly used for quantitative analysis of gene expression, validating DNA microarray
results, and quantifying viral, bacterial, or fungal loads, just to name a few.
Before we get into the types of qPCR.
We first will need to know the instrument to use. In order to get reliable and accurate results from your
qPCR, proper instrumentation is required to provide energy and to detect fluorescence
signals. There are three common ways to provide energy: lamps, LEDs, and lasers. Photodetectors
are used to collect data by only allowing passage of desired wavelengths. Other than
the additional photodetectors, a qPCR machine works very much like a regular thermal cycler
used for PCR amplification. Since qPCR offers real-time output, a special computer software
is needed to analyze the data as it collects. As data is collected, the software shows an
amplification plot which is a log graph with ∆Rn plotted against the cycle number. ∆Rn or the fluoresence signal
is calculated using the equation Rnf – Rnb, where Rnf is the fluorescence emission
at any given point and Rnb is the baseline fluorescence emission. The Ct value is the
number at which ∆Rn of a given sample crosses the threshold value.
The Ct values are inversely correlated to the amount of nucleic acid in your sample.
For example, a lower Ct value indicates higher amounts of the targeted nucleic acid, while
higher Ct values mean that you have very little starting material for amplification.
One type of qPCR is SYBR Green based method, which depends on the intercalating dye, SYBR
Green I. The SYBR Green dye emits a fluorescent signal that is detected by the thermal cycler
when it binds to the double-stranded DNA. In the beginning of the next PCR cycle, the
fluorescent signal is decreased when the double stranded DNA is denatured; therefore the fluorescent signal
of the sample is collected at the end of the elongation step of each PCR cycle. A next
generation DNA binding dye is the EvaGreen dyes, which provide brighter signal than
SYBR Green I dyes. Due to the chemistry of intercalating dyes,
users are unable to distinguish between the desired products and non-specific products
during the PCR cycling stage. The melting curve analysis enables the user to distinguish the
amplified products through plotting fluorescence as a function of temperature. As DNA is heated,
it weakens and eventually breaks the hydrogen bond that holds the two single-stranded DNA
molecules together; this process is called DNA melting or denaturation. Melting
temperature or Tm is defined as the temperature at which the DNA molecule is in mid-transition
between the fully coiled and fully denatured states. For SYBR Green I or EvaGreen based
qPCR’s, the polymerases used must be thermostable and resistant to inhibitors, such as Tfl,
Pwo, Tth, and Pfu DNA Polymerases. The second type of qPCR is called the Taqman
probe method. This method uses 5’-3’ exonuclease activity of the Taq DNA Polymerase which
is paired with special probes that are labeled at both ends (one with fluorescent reporter
dye and the other with a quencher dye). The purpose of having special probes is to prevent binding dyes attaching to non-specific amplicons.
Taqman Probes bind to target sequences on the amplicon. The proximity of two dyes
inhibits the reporter from emitting fluorescence. The Taq polymerase has 5’-3’ exonuclease
activity, allowing cleavage of the 5’ terminal nucleotide. When the polymerase encounters
the probe, it will displace and cleave the 5’ reporter dye. This allows the reporter dye to emit fluoresence since it is no longer being quenched.
The quenching and fluorescence of the probes are explained by two mechanisms: Fluorescence
resonance energy transfer or FRET quenching and non-FRET static quenching. In FRET quenching,
when the donor fluorophore is excited by a photon, it transfers the energy to the acceptor
fluorophore through dipole-dipole interactions. In non-FRET static quenching, the reporter
and quencher are close in proximity. In qPCR, the data is difficult to interpret if both
fluorophores emit fluorescence; therefore quencher dyes in TaqMan probes are usually
dark and do not emit their own fluorescence. The physical interaction between the reporter
and quencher brings the reporter down to a ground state which will quench the reporters
fluorescence. There are several different types of reporter
dyes in TaqMan probes, such as FAM, TET, and JOE and there are different types of quencher
dyes, such as TAMRA, BHQ, and MGB. When choosing the quencher dye that pairs with the reporter,
one must consider both FRET and non-FRET static quenching. In FRET quenching, it is important for the
emission spectrum of the reporter to overlap with the absorbance spectrum of the quencher
and in non-FRET static quenching, the structure of dyes is more important.
One of the major differences between the SYBR Green and TaqMan methods is the use of special
hydrolysis probes in the reaction. Special probes from the TaqMan method only anneal to target
amplicons, decreasing the chance that non-specific and contaminated products in PCR are interpreted
as the target amplicon. By incorporating different fluorophores into hydrolysis probes, different
reporter signals may be detected by the thermal cycler. Therefore this allows the ability to
monitor different amplicons in a single reaction simultaneously, a process called multiplex reaction.
The SYBR Green method is more readily available and less expensive. However, a disadvantage
of SYBR is that the fluorescent dye can bind to both target amplicon and other non-specific
sequences, so fluorescence cannot be distinguished properly from the amplification plot.
At abm, we offer a wide selection of EvaGreen and TaqProbe qPCR MasterMixes. If your starting
material is from cells, be sure to check out our ExCellenCT One-step EvaGreen qRT-PCR MasterMix!
For more information on qPCR principles, please check out our knowledge base.
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