Polymerase Chain reaction, Real-time PCR and Digital PCR

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Polymerase Chain Reaction (PCR)

PCR (Polymerase Chain Reaction) is a key tool that changed the field of molecular biology. This technique has become an integral part of other workflows such as cloning, gene expression analysis, genotyping, sequencing, and several others. PCR has been critical in the evolution of biological applications as it allows amplification of very limited biological samples for use in environmental, clinical diagnostics & forensic studies. The typical PCR reaction (also called end-point PCR) yields qualitative data.

Solutions from

Advanced PCR technologies more accurate and sensitive for target quantitation are:

End Point PCR
Real Time PCR
Digital PCR
Common Equipment

PCR is a repeated process of thermal cycling consisting of 3 key steps:

A typical PCR reaction consists of 35-40 cycles per run. Each PCR cycle results in a doubling of the initial product such that 2n copies of products are generated after ‘n’ cycles. Thermal cyclers, dNTPs and DNA primers specific for region being targeted along with optimal plastics and thermostable DNA polymerases (Enzymes) are key requirements for a successful PCR run. At the end of the PCR run the products can be quantitated spectrophotometrically (A260/A280 nm) measurement and visualized on an agarose gel by gel electrophoresis. A typical PCR workflow follows:

PCR (End-point PCR) workflow

Nucleic acid isolation and quantitation Reverse Transcription and cDNA synthesis (for RNA Templates)Optimization of PCR primer design Reaction set up & Thermal cycling Gel electrophoresis, visualization and data analysis

Direct PCR refers to use of biological sample directly for PCR without nucleic acid isolation Solutions for Direct PCR

Reverse Transcription and cDNA synthesis

RNA cannot be used directly as a template for PCR or Real-Time PCR. The Taq polymerases employed during PCR amplification are DNA template dependent DNA polymerases. Hence, when working with RNA an additional step, Reverse Transcription (RT) is necessary to generate cDNA (complementary to RNA sequence).

Reverse transcriptase’s are enzymes isolated from viruses with a RNA genome. These viruses utilize reverse transcriptase’s to generate cDNA in the process to replicate and re-create more viral particles in the host they attack. Typically RT used for molecular biology applications have been obtained from Mouse Murine Leukemia virus (M-MLV), Avian Myeloblastosis Virus (AMV), or are derivatives of these enzymes. To note, reverse transcriptase enzyme is also found in retroviruses and use for integration of cDNA during integration into the host genome.

To set up the RT reaction, RNA of good quality (free of gDNA) is necessary. Use of DNase I enzymes such as Turbo DNA-free or DNase I should be employed. RNA is combined with dNTP’s and requisite buffers (SuperScript IV VILO Master Mix) and RT enzyme (at 50-55°C) to generate the cDNA. cDNA can be measured using spectrophotometer or a Qubit fluorimeter.

RT reaction can be classified as:

One step RT-PCR : RT and PCR amplification in a single tube using SuperScript IV One-Step RT-PCR System
Two step RT-PCR: Synthesis and purification of cDNA, which is used as template for PCR amplification step

(Superscript IV Reverse Transcriptase + SuperScript IV VILO Master Mix)

Reaction set up and thermal cycling

PCR reaction set up and choice of reagents depends on the length of product to be generated (amplicon size), AT or GC content of the DNA being amplified and downstream applications. Hence, different formulations of buffers, dNTPs, primers and annealing parameters need to be tested. A wide selection of thermostable enzymes for thermal cycling can be employed. Notable among them, AmpliTaq and AmpliTaq Gold, Invitrogen Platinum Taq, AccuPrime, and Platinum SuperFi DNA polymerases have been used and cited often.

The following tool will enable selection of the right buffer and enzyme mix: Selection Tool

Protocol support is available for various PCR applications at: Protocols

Choice of the right plastics is critical. Schema of a MicoAmp plastic plate is shown below.

More information on PCR plastics: Brochure

Thermal cyclers

	ProFlex PCR system	SimpliAmp™ Thermal Cycler	Veriti™ 96-Well Thermal Cycler	Automated Thermal Cycler (ATC)

Catalog Number	4484075	A24811	4375786	A31487
Plastics compatibility	Chart Online Guide
Sample throughput	480,000 reactions	96 reactions	384 reactions	384 reactions
Max block ramp rate	6.0°C/sec	4.0°C/sec	5.0°C/sec	3.5°C/sec
Block formats (optimization of temperature for all 3 steps of the PCR workflow)	3x 32 well 0.2 mL (2-zone VeriFlex block) 96-well 0.2mL (6-zone VeriFlex block) 2x 96-well 0.2 mL 2x flat block 2x 384-well 0.02 mL	96-well 0.2 mL (3-zone VeriFlex block)	96-well 0.2 mL (6-zone VeriFlex block) Fast 96-well 0.1 mL 384-well 0.02 mL 60-well 0.5 mL	96-well 0.2 mL Compatible with full- or semi-skirted plates 384-well 0.02 mL Ideal for automation solutions
Application notes and brochures	Thermal cycling guide –VeriFlex technology

Real-Time PCR (qPCR)

Real-Time PCR (qPCR) is a quantitative tool allowing measurement of product generation as fluorophore intensity after each thermal cycling step. Hence data is captured in real-time. qPCR is highly sensitive and utilized in many clinical diagnostic, forensic and genotyping applications.

The key differences between end-point PCR and Real-Time PCR are as tabulated below:

End-point PCR	Real-Time PCR
Product visualized after complete run at the plateau phase of amplification	Product synthesis monitored after each PCR cycling step in real time and quantified in the exponential phase of amplification
Qualitative and semi- quantitative	Quantitative and used for copy number studies and Gene expression studies. High accurate quantitation of target in sample.
Lower dynamic range	Increased dynamic range of detection
Difficult to multiplex (have more than one DNA target per reaction)	Easy to multiplex
Not very sensitive- use of dyes such as Ethidium bromide	Highly sensitive due to use of high quantum efficiency fluorescent dyes allowing product quantitation after each cycle
Multiple steps and clean up in the workflow	Amplification and detection happens in a single tube, with no post-PCR steps

Ten common pitfalls to avoid in a Real-Time PCR experiment.

Nucleic acid isolation and quantitation Generate cDNA using Reverse- Transcriptase’s and quantitate Real-Time assay selection Thermal cycling and Data Capture Data Analysis

Nucleic acid isolation and quantitation

Real-Time PCR requires DNA/cDNA samples free of any inhibitors that can affect the efficiency of the PCR or quench fluorescence. Hence, special considerations are required during RNA/DNA preparation:

With blood or plant samples it is necessary to remove Haeme or chlorophyll from the sample as they can inhibit fluorescence.
During RNA isolation, all genomic DNA should be removed (Turbo DNA-free or DNAse I)
Washing steps in NA isolation procedures should be strictly followed to ensure no carryover of chaotropic salts, detergents, EDTA etc., into the Real-Time PCR reaction
Use of nuclease-control is critical to prevent false negatives/positive results (See Power of 8)

Common considerations for RNA isolation for Real-Time PCR

More detailed workflow on NA isolation

Generate cDNA using Reverse- Transcriptase’s and quantitate

RT reaction can be classified as:

a. One step RT-qPCR: RT and PCR amplification in a single tube using Verso 1-step RT-qPCR Kit or EXPRESS One-Step SYBR™ GreenER™ Kit

b. Two step RT-qPCR: Generation and purification of cDNA, which is used as template for PCR amplification step

(SuperScript IV VILO cDNA synthesis kit or SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme

Maxima First Strand cDNA Synthesis Kit for RT-qPCR)

Special kits for use with non-coding or miRNA are: TaqPath™ 1-Step RT-qPCR Master Mix, CG

Real-Time assay selection

Assay Selection

The two key chemistries used for Real-Time PCR are the SYBR Green (dsDNA dye binding) and the TaqMan Probe based chemistry.

The TaqMan chemistry incorporates the principle of FRET (Fluorescence Resonance Energy Transfer). During the annealing step of the PCR, a probe specific for the target DNA binds between the two end primers. This probe is designed with a Fluor and a quencher on opposite ends. When the probe is intact, the Fluorophore and Quencher juxtapose each other transferring energy from the fluorophore to the quencher resulting in no fluorescent signal. Newer TaqMan probes incorporate a Non-fluorescing quencher and a Minor Groove Binder molecule to stabilize binding of probe to template requiring smaller size probes 25-30 bases.
During the extension step, the Taq DNA polymerase cleaves the probe releasing the fluorescent molecule into solution- emitting fluorescence. The fluorescence emitted is proportional to the amount of target products generated each cycle. Fluorescence increases after each cycle. (See Figure TaqMan Probe Chemistry using FRET).
Real Time PCR applications

TaqMan vs. SYBR Green Chemistry

Brochure

A Comparison of TaqMan and SYBR chemistries

Criteria	TaqMan Chemistry	SYBR Green Chemistry
	Probe based	Dye binding to dsDNA
Specificity	+++	+
Sensitivity	+++	+/+++
Reproducibility	+++	+/+++
Multiplexing	Yes	No
Ready-made Solutions	Yes	No
Optimization	No	Yes
Cost per assay	++	+

Applications
Gene expression quantitation, SNP genotyping, MicroRNA, Copy number Variation, Pathway Analysis, Digital PCR	Yes	No
Pathogen Detection	Yes	Yes
ChiP	Yes	Yes

Melt Curve Analysis

For SYBR green analysis, it is necessary to optimize the reaction and ensure an unique product is generated after the reaction. The melt curve and its derivative plots allow analysis of products after thermal cycling. A single peak with no extraneous products depicts a successful SYBR green experiment.

Other considerations for SYBR green

It is important in SYBR green optimization that the forward and reverse primers do not have too much cross-homology as this can result in primer-dimers which will bind the dye. Sometimes the non-template control (NTC) can show extra peaks in a melt curve if primer: template ratio’s are kept very high.

TaqMan assays: Infographic

Ask TaqMan for technical support on TaqMan assays

Real time PCR (qPCR) Buffer, Plastics and Reagents

Use of Optimized buffers tested for various applications including Gene expression, Genotyping, small RNA research and Clinical diagnostics is critical for a successful qPCR run. Whether using TaqMan chemistry or SYBR green various Master mixes with ideal formulations of thermostable Taq polymerase, Mg++ salts, dNTP’s are available as tabulated below:

Download Brochure

Chemistry	Application	Start with..
Applied Biosystems™ TaqMan™*	Gene Expression	TaqMan™ Fast Advanced Master Mix for 2-step TaqMan™ Fast Virus Master Mix for 1-step virus detection and gene expression
Applied Biosystems™ TaqMan™*	Pathogen Detection	TaqMan™ Environmental Master Mix 2.0 Environmental and Food samples
Applied Biosystems™ TaqPath™** (Clinical-grade TaqMan)	Gene Expression	TaqPath™ qPCR Master Mix for 2-step TaqPath™ 1-step RT-qPCR Master Mix for 1-step TaqPath™ 1-step Multiplex Master Mix for 1-step & multiplex
Applied Biosystems™ TaqPath™** (Clinical-grade TaqMan)	Genotyping Copy number variation (CNV) (Fast and Standard mode)	TaqPath™ ProAmp Master Mix For human or animal samples
Applied Biosystems™ SYBR™	Gene Expression	Power Up SYBR™ Green Master Mix

Thermal cycling and Data Capture

Real-Time PCR instruments

Real-Time PCR instruments consist of three main components: thermal cycling component, the optics to capture fluorescence after each cycle and interpretation of the data based on the application. Applied Biosystems is a pioneer in real-time PCR and our most quoted systems are tabulated below:

7500 Real-Time PCR System (Catalog: 4351104)	Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument (Catalog: 4406984)	StepOne™ Real-Time PCR System (Catalog:4376357)
StepOne Plus™ Real-Time PCR System (Catalog:4376599)	SureTect™ Real-Time PCR System (Catalog:PT0800)	ViiA™ 7 Real-Time PCR System with 384-Well Block (Catalog: 4453536)
Other Applications: Pharmaceuticals Human identification (HID) for Forensics

The plastic required for various instruments is variable: Choose the correct real-time plastics for your experiment here: Real-Time PCR plastics

Comparisons of newer Real-Time PCR instruments

	QuantStudio™ 3 System Real-Time PCR	QuantStudio™ 5 System Real-Time PCR	QuantStudio™ 6 Flex System Real-Time PCR	QuantStudio™ 7 Flex System Real-Time PCR	QuantStudio™ 12K Flex System (Real-time & Digital PCR)	QuantStudio™ 3D System Digital PCR only
Colors	4 colors	5 or 6 colors	5 colors	6 colors	6 colors	2 colors
Block formats	0.2 mL block (96 reactions) 0.1 mL block (96 reactions)	0.2 mL block (96 reactions) 0.1 mL block (96 reactions)	96-well 96-well Fast 384-well	96-well 96-well Fast 384-well TaqMan Array card (384 well microfluidic card)	96-well 96-well Fast 384-well TaqMan Array card (384 well microfluidic card) Open Array plates	20,000 partitions per chip
Veriflex control	3 zones	6 zones (96-well blocks only)	Not applicable	Not applicable	Not applicable	Not applicable
Applications	Gene expression miRNA profiling SNP genotyping Copy number variation Pathogen detection HRM*	Gene expression miRNA profiling SNP genotyping Copy number variation Pathogen detection HRM* Protein Thermal shift	Gene expression miRNA profiling SNP genotyping Copy number variation Pathogen detection HRM* Protein Thermal shift	Gene expression miRNA profiling SNP genotyping Copy number variation Pathogen detection HRM* Protein Thermal shift	Gene expression miRNA profiling SNP genotyping Copy number variation Pathogen detection HRM* Protein Thermal shift Pharmacogenomics Digital PCR	Copy number variation Pathogen detection Protein Thermal shift Digital PCR Quantification of molecular standards Load determination

Data Analysis

From Fluorescence to Results

In Real-Time PCR experiments, the raw fluorescence collected after each cycle is
corrected against an internal reference dye (ROX) present in the reaction buffer. This normalized fluorescence (called Rn) is plotted against Cycle number.

Typical quantitation experiments measure Relative, Absolute levels of the target gene in the sample or copy number variations of genomic DNA in a sample. Occasionally presence/absence experiments (Example: viral or infectious agent presence) are deployed without quantifying actual target. It is necessary for the accuracy of measurements that all targets are amplified at a high PCR efficiency (80-100%).
More info here: Understanding Ct

Nomenclature

Baseline measurements are used to remove an extraneous fluorescence or noise in the system.
Ct values are inversely proportional to target abundance. Hence, assays for higher copies of target molecules will
cross the threshold at lower cycle numbers (lower Ct). Typically, 40 cycles of thermal cycling are sufficient as even one
copy of target can ideally be measured before this cycle number. Read more about Ct values .

Data Analysis Software

Multiple software are available for Real-Time data analysis.
Instrument software & additional software
Gene Expression, Genotyping software and methods below:

Relative Quantitation (RQ)

Choosing a Normalizer/Endogenous control

Typically used for studies of Gene Expression, Relative quantitation reports target gene abundance as a ratio (fold measurement) in samples under different experimental conditions. Accuracy of these measurements hinge on the choice of the right endogenous controls.

Steps in RQ experiment are:
1) Choose 3-4 representatives for each distinct sample type (treatment, tissue source, time point, etc.) 18s, b-actin, Cyclophilin, HPRT, PGK1, GAPDH
2) Isolate total RNA using a sample prep method appropriate for your sample type(s)
3) DNase-treat to remove any genomic DNA contamination
4) Quantify
5) Reverse Transcribe equal mass amounts of RNA for each sample for cDNA generation
6) Test an equal volume of each cDNA in real-time using the candidate normalizer gene(s)
7) Assess the variability in Ct among the various sample types

TaqMan endogenous gene choices are available for Human, Rat, mouse studies

Relative quantitation (∆∆Ct method)

	Condition 1 (Tumor) Ct value	Condition 2 (Normal) Ct value
Target gene	21	24
Endogenous control (18s, actin, Cyclophilin, HPRT)	18	19
∆Ct	21-18 = 3	24-19 =5
∆∆Ct	3-5 = -2

∆Ct = Ct (target)- Ct (control)
∆∆Ct = ∆Ct condition 1- ∆Ct condition 2
2- (∆∆Ct )= Target gene is 4 fold higher in condition 1 than 2
Here Normal sample is called Reference or Calibrator

Absolute quantitation

Absolute quantitation is typically used in clinical samples or GMO testing. Here absolute copy number of a virus or a target is measured. This measurement correlates the Ct value of a target gene in the sample against a standard curve plot (Ct values of 10-fold dilutions of target vs. Log copy number).

Pictorial depiction of Absolute quantitation:

For viruses and other pre-generated standards visit: Acrometrix Standards

Gene Expression, Genotyping and Copy Number variations

Gene expression and Genotyping workflows are discussed in detail:

TaqMan sources for Copy number variation

Real-Time PCR is used for multiple applications such as:

Digital PCR

Digital PCR is a novel approach to exact copy number (quantification) of a target in the biological sample. This technique is useful for analysis of rare genes, targets and applications such as viral detection, GMO detection and liquid biopsy. The technique utilizes the TaqMan real-time PCR chemistry along with partitioning of input DNA or cDNA. The input material is dispersed into a nano-fluidic chip containing thousands of wells. The resulting partitioning results in some wells containing the input materials and many others with no input. Wells which received multiple input target copies are corrected using a Poisson model of distribution.

Digital PCR circumvents some rate limiting steps with quantitative real-time PCR, namely:

No reliance on copy number standards
Easy precision measurements by duplication of samples across 2 or more chips
Dilution of any inhibitors, so ideal for samples containing PCR inhibitors
Linear amplification with easy correlation of target abundance

Ref: QuantStudio use of Poisson distribution
Popular Products:

Applications of Digital PCR

Digital PCR can complement the Real-Time PCR for use in a variety of applications. This technology is especially adapted for rare mutation detection in a sample, work with viral load quantitation, copy number variation, GMO detection and low level pathogen
Detection. Due to high level of partitioning, negative effects due to presence of inhibitors is diluted
and larger sample volumes can be deployed.

Copy Number variation(CNV) CNV is an important tool to look at genomic alterations and changes. These can be insertions, deletions, translocations. In cancers, Loss of heterozygosity (LOH) and accumulation of mutations is common. Digital PCR with absolute quantitation is a very useful tool for this application.	Rare mutation detection in CfDNA Liquid biopsy can provide a glimpse into disease etiology especially for cancers. Identification of rare mutant alleles involved in tumor maintenance and studies of alleles during therapy can benefit from such studies. Digital PCR with TaqMan Liquid Biopsy dPCR assays are now available for most common cancer related mutations in genes such as EGFR, BRAF, KRAS, PIK3CA, JAK2, and others Flyer	Viral Load quantitation Determination of viral load is critical to assess success of therapeutic interventions. In real-time PCR (qPCR) this requires absolute quantitation using standards of known concentration and the requirement to match identical PCR efficiency, concentrations of samples, instrument calibrations etc., Digital PCR allows for absolute quantitation without need of standards etc.,
Next generation sequencing (NGS) library quantitation Quantification of the amplified libraries is a key step in NGS workflows. Digital PCR with appropriate Taqman assays covering the adapters used for amplification are used here.	Gene Expression Quantification of mRNA transcripts or gene expression benefits from use of digital PCR specifically for situations where differences in gene expression being interrogated is less than two-fold.	Pathogen detection Identification of viruses and pathogens in food an water that can cause illness. Especially useful if low level of pathogens.
GMO detection Food testing to identify genetically modified organisms (GMO) is becoming mandatory in many countries. Real-Time PCR has been used extensively for gene expression, however, identification of low copies in presence of inhibitors such as polyphenols etc., can be challenging.	Reference & Standard Quantification While digital PCR does not require reference standards, it can be used as a tool when generating such materials for other experiments. As a tool used for standard and reference generation, the ease of use means that several references can be deployed to suit the complex biology required by a given experiment.

Digital PCR data analysis

The data from the ™ QuantStudio™ 3D Digital PCR instrument is analyzed with a special software- QuantStudio™ 3D Analysis Suite™ Cloud Software. Since digital PCR is based on partitioning, the data from a single chip (of 20K wells) is collected together in a single experiment data (.eds) file. This file is a composite of the processed imaging data + all results from preliminary analysis. The data can be interpreted to generate relative quantification (RQ) or absolute quantification results. Data can be stored in the cloud or on a local network server depending on the user’s needs.

Important Features: