Attention:

Certain features of Sigma-Aldrich.com will be down for maintenance the evening of Friday August 18th starting at 8:00 pm CDT until Saturday August 19th at 12:01 pm CDT.   Please note that you still have telephone and email access to our local offices. We apologize for any inconvenience.

Life Science Quarterly

Sybr Green Feature Aticle

 Life Science
Quarterly
 A TECHNICAL APPLICATION NEWSLETTER VOL 3   ISSUE 1   Winter 2002

 Molecular Biology Feature Article

SYBR® Green JumpStartTM Taq ReadyMixTM: Lights Your Way To Quantitative PCR

By Erinn von Rein 
Sigma-Aldrich Corporation, St. Louis, MO USA

Introduction

Quantitative PCR (qPCR) has developed into a popular technique since being pioneered by Higuchi et al. in the early 1990's. 1 The technique was established to quantify the gene copy number of DNA or the number of transcribed RNA copies through continuous monitoring of fluorescent signal. During PCR amplification, an introduced fluorescent reporter generates a signal that is proportional to the DNA concentration.

Quantitative PCR is both highly sensitive and precise. Two basic chemistries, which are the keys to making this technique extremely sensitive, have been used in all qPCR systems. The first, a probe-based system, utilizes a sequence-specific probe for quantitation of the template of interest. In the second method, intercalating dyes fluoresce only when bound to double-stranded products generated by PCR. The design of highly specific primers and optimized reagents insure sensitive quantitation. Quantitative PCR relies on identification of the first cycle that gives a signal over the background, and calculation of this threshold cycle (C T ) makes quantitation much more precise than end-point analyses. The sensitivity and precision of qPCR allows this technology to be compatible in innumerable applications. 2 Gene detection, measurement of transcription levels, and allelic genotyping are some of the applications for which qPCR has become a vital technique.

This report focuses on the use of SYBR Green I dye with quantitative PCR. Accurate quantitation can be obtained over a large range of initial template amounts if conditions are optimal. SYBR Green JumpStart Taq ReadyMix has been shown to be linear over a 108-fold range of template concentrations, sensitive down to 10 copies, and generally performs as well as competitive mixes while providing the convenience of a ReadyMix (master mix). Inclusion of anti-Taq antibody suppresses nonspecific product and allows SYBR Green I dye to intercalate solely into the template of interest. 

Materials and Methods

All materials were supplied by Sigma-Aldrich Corporation (St. Louis, MO) unless otherwise stated. SYBR Green I dye was received from Molecular Probes (Eugene, OR) and pBAC-2cp was obtained from Novagen Corporation (Madison, WI).

DNA Templates

pBAC-2cp and lambda DNA were initially quantified spectrophotometrically at 260 nm. 

Quantitative PCR Conditions

Experiments were conducted in the ABI PRISMTM 7700 Sequence Detection System (Perkin Elmer/Applied Biosystems, Foster City, CA). SYBR Green JumpStart Taq ReadyMix (2X; Product Code S 4438) contains: 20 mM Tris-HCl (pH 8.3), 100 mM KCl, 7 mM MgCl2, 0.4 mM each dNTP (dATP, dCTP, dGTP, TTP), stabilizers, 0.05 unit/ml Taq DNA Polymerase, JumpStart antibody, and SYBR Green I dye. All reactions were performed in 50-ml reaction volumes that contained 1X SYBR Green JumpStart Taq ReadyMix, 1X internal reference dye, 400 nM each of plasmid-specific primers, and various concentrations of template DNA.

Sensitivity experiments were performed using pBAC-2cp as a template, and plasmid-specific primers designed to produce a 311 bp amplicon. The PCR was initiated with a 1-minute denaturation step at 95 °C. Initial denaturation was followed by 40 cycles at 95 °C for 15 seconds, 1-minute annealing at 60 °C, and 1-minute extension at 72 °C. Cycling was followed by a 4 °C hold. The template was diluted ten-fold past zero copies. Analyses of data were accomplished using the ABI PRISM 7700 Sequence Detection Software. In the internal reference dye experiments, various dyes were titrated into reactions to find an optimal dye concentration. PCR was performed using lambda DNA dilutions with specific primers that produced a 201 bp amplicon. Thermal cycling conditions were as previously described.

Modified nucleotide experiments were run using SYBR Green JumpStart Taq ReadyMix, 10 ng of cDNA from HeLa cells, and gene-specific primers targeted to cyclin activated kinase (CAK1) associated protein at a final concentration of 400 mM. Modified nucleotides were used at concentrations ranging from 0-400 mM with standard dNTPs supplementing the modified nucleotides as needed to maintain a total dNTP concentration of 800 mM in each reaction. PCR conditions began with a 94 °C denaturation step for 1 minute. Initial denaturation was followed by 30 cycles at 94 °C for 30 seconds, 58 °C for 30 seconds and 68 °C for 4 minutes. A 68 °C hold was performed for 7 minutes following cycling. 

Results and Discussion

SYBR Green JumpStart Taq ReadyMix produces accurate results over a large linear range

The linear range over which SYBR Green JumpStart Taq ReadyMix is useful was explored in a series of experiments in which several different templates were analyzed. A representative experiment is shown in Figure 1 , and the analysis of this set of reactions is in  Figure 2 . SYBR Green JumpStart Taq ReadyMix has been shown to produce results that are linear over 8 logs of initial template concentrations. In addition the data show that the use of SYBR Green JumpStart Taq ReadyMix allows accurate quantitation of as few as ten copies. Results are reproducible over a wide-range of starting template amounts.

Low sensitivity is often a major drawback when using SYBR Green I dye in quantitative PCR reactions. Non-specific products formed in the PCR reaction contribute to the fluorescent signal and produce CTs that appear much earlier in the cycling process than should actually be seen. The inclusion of a hot start mechanism in the qPCR reduces nonspecific product information. Figure 3 illustrates how the inclusion of the anti-Taq antibody in the SYBR Green JumpStart Taq ReadyMix insures clean, single-band amplification.

Internal reference dye added to SYBR Green ReadyMix produces less error in qPCR reactions. An internal reference dye is important in quantitative PCR for the normalization of reporter dye signal. It becomes increasingly important when two or more reporter dyes are present in the PCR reaction. Normalization produces less error in the final data analysis and keeps high CTs more reproducible.

Sigma's reference dye is included as a separate component in the SYBR Green qPCR system, allowing the user to decide when to add the standard. In determining the type and concentration of dye to be used with the system, three criteria were set. Initially, the dye had to be soluble in water, give an appropriate number of relative fluorescence units (RFUs) without affecting the reporter signal (SYBR Green I dye in this case), and most importantly would not poison the PCR reaction. Several dyes were screened to provide the needed sensitivity and solubility. A series of experiments were run in qPCR with various dye concentrations. The dye chosen supports all of the previously stated requirements. With an emission maximum of 605 nm the dye is far removed from the reporter signal and will not interfere with its fluorescence. 

Too much internal reference dye skewed the CTs while too little provided no normalization effect. The final recommended dye concentration uses the minimum amount of dye needed to give a reasonable normalization signal ( Figure 4 ).

Effect of modified nucleotides can be quantitated using SYBR Green I dye

One use of qPCR is exemplified in the optimization of PCR yield when modified nucleotides are included in the reaction. At Sigma-Aldrich, modified nucleotides are used to generate inserts for cloning via the ExoCloneTM technology. Although modified nucleotides are crucial for this cloning technique, they can have an inhibitory effect on PCR. A modified nucleotide concentration that is too high will reduce the efficiency of the PCR reaction.

The total NTP concentration was held constant throughout all the concentration of modified dNTPs increased, the concentration of standard dNTPs decreased. As seen in  Figure 5 , modified dNTPs eventually poison PCR, but moderate amounts allow reasonable incorporation without affecting yield. From this qPCR amplification plot, a concentration of 150 mM was chosen as the concentration that supported incorporation and PCR yield. At this concentration a functional amount of modified nucleotides was incorporated while PCR yield and efficiency were maintained. 

Summary

Advantages of SYBR Green JumpStart Taq ReadyMix can be readily seen when performing high-throughput qPCR. Its ease of use combined with high sensitivity and broad-linear range make it a good choice for qPCR. Addition of an internal reference dye makes the ReadyMix increasingly accurate by signal normalization over multiple cycles and conditions. Furthermore, SYBR Green I dye can be used to quantify a wide variety of templates in many applications. 

Acknowledgments

The author would like to thank Sigma-Aldrich R&D, especially Ron Hernan, for providing the data on modified nucleotides. 

References

1. Higuchi, R., Dollinger, G., Walsh, P. S. and Griffith, R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY), 10, 413-417 (1992). 

2. Bustin, S.A., Absolute quantification of mRNA using real-time reverse transcription polymerase. Journal of Molecular Endocrinology, 25, 169-193 (2000). 

Purchase of these products is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. These products are sold under licensing arrangements with F. Hoffmann-LaRoche Ltd., Roche Molecular System, Inc. and the Perkin-Elmer Corporation. 

About the Author

Erinn von Rein, B.S., is an Associate Scientist in the Biotechnology R&D Department of Sigma-Aldrich, St. Louis, MO.
 

 
ORDERING INFORMATION
Product Code Product Name Unit
S4438 SYBR® Green JumpStartTM Taq ReadyMixTM 100 reactions


500 reactions

For more information, see Frequently Asked Questions

   
 
   
 

Back to Contents