( Faye Boeckman Marni Brisson...)Real-Time PCR: General Considerations, Rev A

Faye Boeckman, Marni Brisson, and Larissa Tan, Bio-Rad Laboratories, Inc., Hercules CA 94547 USA

General Information
The polymerase chain reaction (PCR) has proven to be a versatile tool in molecular biology. The use of this technique has generated unprecedented advances in gene discovery, diagnostics, and gene expression analysis. In addition, new techniques that build on PCR have further expanded its range of scientific applications.

Real-time PCR is a powerful advancement of the basic PCR technique. Through the use of appropriate fluorescent detection strategies in conjunction with proper instrumentation, the starting amount of nucleic acid in the reaction can be quantitated. Quantitation is achieved by measuring the increase in fluorescence during the exponential phase of PCR. Applications of real-time PCR include measurements of viral load, gene expression studies, clinical diagnostics, and pathogen detection.

Although the performance of PCR in more routine molecular biology applications can be relatively straightforward to optimize, several parameters must be evaluated and optimized independently to achieve the maximum potential of real-time PCR. The factors that affect real-time PCR fall into 3 categories. These are general laboratory practices, template and primer design, and reaction components and conditions. When determining which conditions to optimize, the ultimate assay goal (i.e., qualitative analysis vs. quantitation) must be considered.

To develop sensitive real-time PCR assays cost effectively, you should design and optimize the primer sets prior to developing the probe. This technical note will guide you in the development of an optimized primer set for a quantitative real-time assay.

Considerations for General PCR Optimization
General Laboratory Practices for Quantitative Real-Time PCR
In general, follow these practices to ensure the highest probability of success:
Wear gloves
Use screwcap tubes
Use aerosol-resistant filter tips
Use calibrated pipets dedicated to PCR
Use PCR-grade water and use only for PCR
Use a no-template control to verify absence of contamination
Prepare reactions in replicate ideally as triplicates

Replicate Quality
To obtain good replicates, a master mix should be prepared with all reaction components including the sample Use a hot-start enzyme to prevent nonspecific amplification during preparation
Make up a master mix with sufficient volume to prepare all replicate samples
Pipet once per well

DNA Source
The source of the template affects the accessibility of the target sequence and must be considered during optimization. It is important to optimize the reaction for the template concentrations that will be used in your experiment.

Genomic DNA (Intact, High Molecular Weight DNA)
Cut with a restriction enzyme that does not cut within the region to be amplified
Boil DNA stock for 10 min and place immediately on ice

Plasmid DNA
If there are problems with amplification, linearize the plasmid with a restriction enzyme that does not cut within the target

cDNA
RNA must be free from genomic DNA contamination treat with RNase-free DNase prior to reverse transcription. It is also helpful to design primers at splice junctions to avoid genomic DNA amplification

Template and Primer Design
Template Design
A successful real-time PCR reaction requires efficient amplification of the product. Both primers and target sequence affect this efficiency. Significant template secondary structure may hinder the primers from annealing and prevent complete product extension by the polymerase. Follow these guidelines:
Amplify a template region of 75150 bp
Avoid secondary structure if possible
Use an annealing temperature above the melting temperature (T_m) for any template secondary structures
Avoid templates with long (>4) repeats of single bases
Maintain a GC content of 5060%
Analyze secondary structure with the DNA mfold server of Dr. Michael Zuker or equivalent program at:
http://bioinfo.math.rpi.edu/~mfold/dna/form1.cgi

Secondary Structure Analysis
To evaluate secondary structure of a product on Dr. Zukers site:
1. Name the sequence example: bactin1
2. Copy the sequence or retype it into the big text box
3. Scroll down the page to the input labeled Folding temperature and enter the annealing temperature of the reaction
4. Scroll down the page to the input labeled Ionic conditions and change the units to mM. Adjust the values to reflect the ionic conditions in the reaction (set [Na⁺] to 50 mM and [Mg²⁺] to 3 mM for most reactions)
5. Scroll down further to enter your e-mail address you will not receive an e-mail message, but the program will not proceed without it
6. Click on the button marked Fold DNA next to the smiley face
7. Now youll get a list of structures ideally you will see only one. Pick a format such as PNG to view the structure.
8. The T_m of the structure, which appears in a separate window (Loop Free-Energy Decomposition), will tell you at what temperature this structure will form.

Primer Design
The goal is to design primers with a T_m higher than the T_m of any of the predicted template secondary structures. This ensures that the majority of possible secondary structures have been unfolded before the primer-annealing step. Follow these parameters when designing primers:
Design primers with a GC content of 5060%
Maintain a melting temperature (T_m) between 50 and 65C
Eliminate secondary structure
Avoid repeats of Gs or Cs longer than 3 bases
Place Gs and Cs on ends of primers
Check sequence of forward and reverse primers to ensure no 3' complementarity (avoids primer-dimer formation)
AdjusT_ment of primer locations outside of the target sequence secondary structure may be required
Verify specificity using sites such as the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/blast/)

Reaction Components and Conditions
Components
Optimization conditions can vary with assay type. Therefore, these conditions should be considered when establishing a new assay:
MgCl₂ concentration (3.06.0 mM)
dNTP concentration (200600 M each dNTP) Increasing the [dNTP] will require an increase in [MgCl₂]
Source and concentration (1.254.5 U/50 l reaction) of Taq DNA polymerase
Primer concentration (100500 nM)
An asymmetric primer concentration may be helpful
Fluorescent probe or intercalation dye concentration

Conditions
Optimization of the following amplification conditions will be required to obtain the maximum efficiency and specificity:
Annealing temperature (50 to 65C) and time (dependent on primer T_m and chemistry)
Extension time (dependent on chemistry and product length)
Denaturation temperature and time (dependent on target sequence)
2-step v. 3-step PCR

Experimental Design and Interpretation of Results
Primer Selection
This section demonstrates the importance of primer optimization using the human cyclophilin 40 gene. Two sets of primers, differing in location, were designed to amplify the same region of the human cyclophilin 40 gene (IMAGE Consortium clone 71154, ATCC). Figure 1 illustrates the location of the primer sets (primer sets A and B use the same forward primer).

Five replicates for a 10x dilution series (10⁷ to 10³ copies) using identical primer concentrations (300 nM/reaction) were performed on the iCycler iQ system. The reaction mixture consisted of custom-made Life Technologies Supermix (Platinum Taq polymerase, 1.25 U, 20 mM Tris, pH 8.4, 3 mM MgCl₂, 0.2 mM of each dNTP, 50 mM KCl). Real-time amplification was detected using the intercalating dye SYBR* Green I (Molecular Probes; 1:75,000 dilution of the 10,000x stock solution).

Optimizing primer location to reduce template secondary structure interference increased both the sensitivity and efficiency of amplification. Using the formula E = (10^-1/slope)-1 to calculate efficiency (E), a reaction with 100% efficiency will generate a slope of -3.32. The amplification plot of the experiment using primer set A generated a slope of -4.53 or 66% efficiency, with a correlation coefficient of 0.995 (Figure 2, upper panel). Moving the reverse primer inwards to the location of primer B shortened the PCR product and eliminated strong secondary structure. This shift in primer location significantly improved the efficiency of the reaction to 99% (slope = -3.35) and a correlation coefficient of 0.999 (Figure 2, lower panel).

MgCl₂ Evaluation
Typically, real-time PCR requires higher concentrations of MgCl₂ for optimal results. To demonstrate this, an analysis of MgCl₂ concentration effects on amplification efficiency was performed. Replicate reactions at 4 MgCl₂ concentrations (1.5, 2.25, 3, and 4 mM) and final primer concentrations of 300 nM each (for the optimized Primer B pair from above) were prepared. Reaction conditions were 1.25 U Platinum Taq DNA Polymerase and 1x PCR buffer (Life Technologies), 0.2 mM each dNTP (Advantage Ultrapure dNTPs, Clontech), and SYBR Green I (Molecular Probes, 1:75,000 dilution of the 10,000x stock solution).

The magnesium concentration had a significant impact on the amplification efficiency of the PCR reactions as demonstrated in Figure 2 and Table 1. The highest PCR efficiency was achieved with a 3 mM MgCl₂ concentration (Table 1). The importance of maintaining high amplification efficiencies is clearly depicted in Figure 3. The threshold cycle was shifted by 4.7 cycles at 10⁴ copies and by 3.6 cycles at 10⁷ copies when the MgCl₂ concentration was increased from 1.5 mM to 3.0 mM. This significant shift in CT was not observed in the 2.25 mM or 4 mM MgCl₂ samples.

PCR efficiency can be affected by numerous factors. In this technical note we have evaluated 2 of these factors secondary structure interference and free magnesium ion concentration. We have clearly demonstrated that optimization of PCR design and reaction conditions on this template has strong effects on the quality of real-time PCR assays.

Once the primer locations and the MgCl₂ concentrations have been evaluated, other reaction components, such as probe concentration and protocol temperatures, may need to be evaluated. For example, a 2-step or 3-step PCR thermal protocol may yield optimal amplification detection, depending on the detection strategy. Although this initial optimization may seem cumbersome, it will result in reaction conditions that are robust and reproducible.

* SYBR is a trademark of Molecular Probes, Inc. Practice of the patented polymerase chain reaction (PCR) process requires a license. The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from PE Corporation. Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents. Some applications may require licenses from other parties.

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