| HOME >> BIOLOGY >> TECHNOLOGY |
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 opti mized 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 tr
anscription. 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 (Tm)
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 Tm 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 Tm higher than the Tm 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 (Tm) 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)
AdjusTment 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 Tm 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 seco ndary 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 i s 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.
back to top
'"/>
Source:
Related biology technology :
1. Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System
2. Monitoring Microbial Populations Using Real-Time qPCR on the MJ Research Opticon 2 System
3. Rapid SNP Genotyping of the Pharmacogenomically Important Allele CYP2D6*4 Using Real-Time qPCR on the DNA Engine Opticon 2 System
4. Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System
5. Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A
6. Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye
7. Quantitation of Lymphangiogenesis Using the iCycler iQ Real-Time PCR Detection System and Scorpions Detection System, Rev A
8. Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A
9. Real-Time Immuno-PCR on the iCycler iQ System, Rev A
10. Real-Time PCR/Melt-Curve Analysis: SNP Detection With FRET, Rev A
11. General Notes on Primer Design in PCR*
| Copyright © 2003-2012 Bio-Medicine. All rights reserved. ABOUT | CONTACT US | DISCLAIMER | PRIVACY POLICY | TERMS AND CONDITIONS |  |