APPLICATION NOTE: Reproducible Multiplex Amplification Data in 24 Minutes

Fast Multiplex qPCR using CleanAmp™ PCR Kit and the Mastercycler® ep realplex4 S qPCR instrument.

By Elena Hidalgo Ashrafi & Natasha Paul, Ph.D.; TriLink BioTechnologies

Introduction

Multiplex real-time PCR (qPCR) is a powerful technique that helps to save time, to conserve limiting samples, and to reduce the cost of genetic analysis. While there are many benefits to multiplex qPCR assays, extensive optimization is often required as the increased number of primer sets per assay can compromise amplification efficiencies. Advances in molecular diagnostics demand shorter assay times which further challenge the generation of replicable and accurate fast mutiplex qPCR results. We demonstrate triplex qPCR by combination of CleanAmp™ dNTPs and a sensitive qPCR instrument.

CleanAmp™ dNTPs provide a universal approach to Hot Start activation in PCR. Simple substitution of CleanAmp™ dNTPs for standard dNTPs in PCR offers the same advantages as more costly Hot Start enzymes. CleanAmp™ dNTPs offer precise control at the start of PCR thermal cycling by blocking nucleotide incorporation until heat activation of the dNTPs (1). The temperature-dependent control of DNA polymerase extension vastly reduces mis-priming, primer dimer formation and other deleterious effects which commonly occur, resulting in optimal performance during multiplex (2) and fast PCR. The CleanAmp™ dNTPs are available in a kit, which includes a DNA polymerase validated for assay success. The CleanAmp™ PCR Kit provides the flexibility for use in a variety of assay formats, including fast-cycling and multiplexed PCR.

The Eppendorf realplex4 S real-time PCR system features a super-fast, Peltier-controlled thermoblock that heats up at 6°C/sec and cools down at 4°C/sec, which allows completion of a standard 40-cycle qPCR reaction in less than one hour. The Peltier units also ensure accurate temperature control as well as high block homogeneity, which are essential to high-quality PCR reactions. The optical module comprises an array of 96 LEDs which are pre-selected and equalized so that they excite all wells uniformly. This setup eliminates the requirement for ROX as a reference dye, thereby making it available for use as a Taqman® dye. This is a must by many conventional qPCR instruments. Realplex4 S also utilizes Photo Multiplier Tube (PMT) for signal detection, which is the most sensitive and affordable detection technique currently available.

To further explore the benefits of the CleanAmp™ PCR Kit and the Eppendorf realplex4 S real-time PCR system, the studies herein feature the combination of these two technologies to achieve a reproducible real-time triplex PCR assay that can be completed in less than 30 minutes.

Methods

The CleanAmp™ dNTP Kit was used to perform a real-time triplex assay in a realplex4 S fast cycling instrument with hydrolysis probe detection. Two experiments were performed which amplified three mouse genomic DNA targets of sizes: 125, 185 and 214 base pairs using the CleanAmp™ PCR Kit. First, simultaneous triplex amplification of all three targets was performed using standard and fast thermal cycling conditions (Figure 1). Second, the three targets were amplified both simultaneously and individually using the fast protocol (Figure 2). All reactions were performed in Eppendorf's real-time PCR tube strips with Masterclear™ Cap Strips using hydrolysis probe detection: 125 bp (6-JOE; 0.2 µM), 185 bp (FAM; 0.05 µM), and 214 bp (ROX; 0.2 µM).

Figure 1. Consistent triplex amplification under standard and fast thermal cycling conditions. Data shown is a 5-fold dilution series of mouse genomic DNA performed in quadruplicate: (A) amplification plots, (B) standard curves and (C) a data compilation table.

Triplex qPCR detection with protocols suited for standard and fast cycling were evaluated. All reactions were set up in quadruplicate and used a series of 5-fold serial dilutions of mouse genomic DNA ranging from 320 pg to 1.0 µg. Protocols for the Standard Thermal Cycling qPCR experiment followed the "multiplexed PCR protocol" from the CleanAmp™ PCR Kit Product Insert (1X PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl and 2.5 mM MgCl2), 0.4 mM CleanAmp™ dNTPs, 0.01 U/µL Taq DNA polymerase, and 0.2 µM primers, 25 µL). The fast cycling multiplexed PCR protocol followed the above "multiplexed PCR protocol" with the following alterations: 70 mM KCl, 4.0 mM MgCl2, 0.6 mM CleanAmp™ dNTPs, 0.33 U/µL U Taq DNA polymerase, primers (0.5 µM for the 125 bp and 185 bp sets and 0.7 µM of the 214 bp primer set) in 15 µL.

Figure 2: Comparison of singleplex and triplex amplification protocols under fast thermal cycling conditions. Data shown is a 4-fold dilution series of mouse genomic DNA performed in quadruplicate: (A) overlaid singleplex and triplex amplification plots with the corresponding standard curves and (B) a data compilation table.

The CleanAmp™ dNTP Kit was then further evaluated in fast cycling conditions for amplification of the same three targets in singleplex and multiplex reaction formats. This assay was set up in triplicate for amplification of 5-fold serial dilutions of mouse genomic DNA ranging from 320 pg to 200 ng. The fast cycling multiplex protocol and thermal cycling conditions as described above were followed. For the singleplex reactions, the "fast thermal cycling protocol" from the CleanAmp™ PCR Kit Product Insert was followed (1x PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl and 4.0 mM MgCl2), 0.4 mM CleanAmp™ dNTPs, 0.33 U/µL Taq DNA polymerase, and 0.5 µM primers, 15 µL).

Results and Conclusion

Constant advances push the limits of PCR by demanding more rapid, higher throughput assays. Fast multiplex qPCR allows for simultaneous analysis of multiple targets, but can be plagued with challenges that hinder its efficiency and the specificity of detection.

When standard and fast cycling protocols for triplex qPCR were compared, results show robust performance for thermal cycling times of 1 h 26 min and 24 min. In both reaction formats, the amplification curves were well spaced with tight replicates. The resultant standard curves were fit with good linearity as evidenced by the R2 values and revealed comparable efficiencies from target to target and assay to assay (Figure 1). Similar results were obtained when the amplification of each target was compared in a singleplex and a triplex reaction under fast cycling conditions (Figure 2A). For each target concentration, the amplification curves for singleplex and triplex reactions coincide as depicted in Figure 2. Consequently, the similar Cq values for each reaction format provided comparable efficiencies as reflected in the Table (Figure 2B). These findings highlight the versatility of the CleanAmp™ PCR Kit and the speed of the Mastercycler® ep realplex4 qPCR instrument, which complement one another to achieve efficient and reproducible triplex amplification data in only 24 minutes.

The combination of the CleanAmp™ PCR Kit and the Mastercycler® ep realplex4 S qPCR instrument provides the ability to perform a triplex PCR reaction in a fast cycling mode maintaining the high amplification efficiencies that can be obtained in standard cycling or in a singleplex.

For more information on the CleanAmp™ PCR Kit or Eppendorf® Mastercycler ep realplex4 S real-time PCR instrument please visit: http://www.trilinkbiotech.com/products/cleanamp/ or www.eppendorf.com/realplex.

References

  1. Koukhareva, I. and Lebedev, A. (2009) 3'-Protected 2'-Deoxynucleoside 5'-Triphosphates as a Tool for Heat-Triggered Activation of Polymerase Chain Reaction. Anal Chem., 81, 4502-4509.
  2. Le, T., Hidalgo Ashrafi, E. and Paul, N. (2009). Enhancing multiplex PCR efficiency using Hot Start dNTPs. BioTechniques. 47, No. 5, 972-973.