HotStart-IT^® Probe qPCR Master Mix with UDG (2X)

HotStart-IT^® Probe qPCR Master Mix with Uracil-DNA Glycosylase (UDG) uses a novel hot start method developed at USB called primer sequestration. With this method, a protein binds and sequesters primers at lower temperatures making them unavailable for use by Taq DNA Polymerase (Fig. 1). Following the initial denaturation step, the protein is inactivated and the primers are released. HotStart-IT Probe qPCR Master Mix with UDG is supplied as a 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl₂, Ultrapure nucleotides with an optimized dUTP to dTTP ratio, and heat-labile UDG for use in real-time quantitative PCR reactions (qPCR) with fluorescent probes. Simply add DNA template, primers, probe(s) and water and the reactions are ready for cycling. A separate tube of ROX passive reference dye (for ABI and Stratagene instruments) is included for added convenience.

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Since the mix contains dUTP and UDG, carry-over contamination prevention can be performed, which is especially important for high-throughput applications⁽¹⁾. A heat-labile version of UDG that can be irreversibly heat-inactivated is used instead of E. coli UDG, which has been shown to exhibit residual activity following PCR reactions⁽²⁾. The mix is formulated for use with fluorescent probes such as TaqMan^® Probes, Molecular Beacons, and others^(3-4). Since fluorescent probes are designed to hybridize to the target of interest, detection specificity is greatly increased relative to nonspecific dsDNA binding dyes such as SYBR^® Green I. The Taq DNA Polymerase used in the master mix has the 5'3' exonuclease activity necessary for efficient removal of the 5'-fluorophore from the 3'-quencher in TaqMan probes. HotStart-IT Probe qPCR Master Mix with UDG has excellent sensitivity as it detects fewer than 10 target copies, performs over a broad, linear dynamic range of 7 to 8 orders of magnitude, and is compatible with a variety of real-time PCR instruments.

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Real-Time PCR Brochure

Convenient
Save time and reduce potential contamination errors by eliminating several pipetting steps. For a 50 μl reaction, simply add 25 μl of master mix to primers and probe(s), DNA template and PCR Qualified H₂O. Reactions can be tailored from 20 μl to 100 μl volumes.

Room temperature reaction assembly is possible because of the hot start feature.

Multiple Platform Compatibility
Specialized buffer with optimal MgCl₂ concentration and dUTP to dTTP ratio performs well on a variety of platforms. Also, the separate tube of ROX Passive Reference Dye allows normalization of well-to-well variations that may occur independent of the reactions (e.g., pipetting errors, detection system limitations, etc.).

Carry-Over Contamination Prevention
Eliminate at least 10⁵ copies of dUTP-containing contaminating templates. The dUTP in the mix ensures that amplicons contain uracil which can be destroyed prior to subsequent amplification reactions by the enzymatic activity of the Uracil-DNA Glycosylase also included in the mix. After the initial denaturation step, the UDG is inactivated, and only the desired target sequences without dUTP are amplified.

Advantage with Heat-Labile UDG
The UDG used in the USB master mix, unlike E. coli UDG used in most other products, is completely and irreversibly heat-inactivated due to the high temperature cycling conditions. This maintains the integrity of the PCR products following reactions which is important if they are to be used in subsequent analyses such as gel electrophoresis, cloning, and/or sequencing.

Novel Hot Start Technology
Since the polymerase is not chemically-inactivated, no extensive heat-activation step is necessary which reduces damage to precious DNA samples.

Higher Specificity, Sensitivity and Broad Dynamic Range
The hot start feature minimizes amplification of non-specific products and primer-dimers. The reaction buffer with optimum MgCl₂ concentration is specially designed for robust probe hybridization and efficient cleavage of TaqMan^® probes. PCR products may be amplified with low background and from low-copy targets with a linear dynamic range of 7 to 8 orders of magnitude (Fig. 2).

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GAPDH Assay using HotStart-IT Probe qPCR Master Mix with dUTP and UDG (PN 75764). Triplicate reactions were performed with a cloned region of the human GAPDH gene as template using an ABI 7500 Fast instrument. A TaqMan® probe with FAM as the reporter fluorophore and BHQ-1® as the quencher was used to detect the 122 bp amplicon. ROX was used as a passive reference dye. The amplification process was linear over eight orders of magnitude (see inset) and a single copy of the target could be efficiently detected. The No Template Control (NTC) reaction generated no measurable fluorescence.

Stable
Repeated freeze-thaw cycles have no observed effect on performance.

Tested User Friendly™ Functional Test:
Real-time PCR reactions were performed on an ABI 7500 Fast Instrument using primers and TaqMan^® probe specific to a 122 bp cloned region of the human GAPDH gene as template. Product specifications require that the correlation coefficient from a linear regression over seven
orders of magnitude (10 to 10⁷ template copies) must be greater than or equal to 0.95.

Additionally, carry-over contamination tests of the UDG activity in the mix were performed. Product specifications require removal of greater than or equal to 10⁵ dUTP-containing template copies per reaction.

HotStart Probe qPCR Master Mix with UDG (2X):
The mix combines USB HotStart-IT Taq DNA Polymerase (with 5'3' exonuclease activity), heat-labile UDG, MgCl₂, and Ultrapure nucleotides with an optimized dUTP to dTTP ratio in a unique buffer formulation. Magnesium and nucleotide concentrations are 6mM and 0.4mM each, respectively.

Components:
HotStart-IT^® Probe qPCR Master Mix with UDG (2X)
  100 reactions 2 x 1.25 ml
  500 reactions 12.5 ml
25mM MgCl₂
ROX Passive Reference Dye
Brief Protocol

References:

1. Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, 125-128.
2. Thornton, C. G., Hartley, J. L., and Rashtchian, A. (1992) BioTechniques 13, 180-184.
3. Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W., and Deetz, K. (1995) PCR Methods Appl. 4, 357-362.
4. Tyagi, S., and Kramer, F. R. (1996) Nat. Biotechnol. 14, 303-308.

Storage:
Shipped on dry ice. Store at -20°C. Mix well prior to use.