KAPA PROBE FAST qPCR Kits have been optimized for versatility and speed – providing fast and reproducible results for genotyping, gene expression analysis, and multiplexing with a single kit.

KAPA PROBE FAST qPCR Kits are designed for high throughput, fast-cycling, real-time PCR using sequence-specific fluorogenic probes. These kits are compatible with all fluorogenic probe-based technologies, including hydrolysis probes (e.g. TaqMan^®) and displacement probes (e.g. molecular beacons).

The KAPA PROBE FAST qPCR Master Mix (2X) is a ready-to-use cocktail containing all components except primers, probe(s) and template for fast cycling probe-based real-time PCR. The 2X Master Mix contains KAPATaq HotStart DNA polymerase, KAPA PROBE FAST qPCR Buffer, dNTPs, MgCl2 and stabilizers.

KAPATaq HotStart DNA Polymerase is an antibody-mediated hot start formulation of KAPATaq DNA polymerase. In the HotStart formulation, the enzyme is combined with a proprietary antibody that inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency.

KAPA PROBE FAST qPCR Kits provide:

Compatibility with all probe-based qPCR applications and instruments
Fast, reproducible, and precise quantification
Discrete clusters in SNP genotyping assays
Multiplex qPCR
Broad dynamic range
Highly stable master mix for high-throughput workflows

Discrete clusters and high call rates for accurate and reproducible allelic discrimination

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All 168 human genomic DNA samples were accurately genotyped using the ABI sequence detection system (SDS) version 2.3 software (autocaller confidence level 95%). A total of 168 human genomic DNA samples were successfully genotyped along with 24 no-template controls using an ATP1B3 SNP genotyping assay on the ABI 7900HT real-time PCR system. Reactions were performed in 5 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10 ng per reaction), 200 nM of each primer and 200 nM of each hydrolysis probe (Allele X – FAM/ BHQ®-1, Allele Y – VIC/ BHQ®-1) using the following standard cycling protocol: 95 ºC for 10 min followed by 40 cycles of 95 ºC, 15 sec; 60 ºC, 60 sec.

Fast, high performance five-color multiplexed qPCR

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Highly reproducible and efficient results for all 5 amplicons across a 5 point dilution series of human genomic DNA were obtained when assayed in penta-plex using a fast cycling protocol. Standard curves were generated using 4-fold dilutions of human genomic DNA (0.39 – 100 ng per reaction) tested in triplicate using the Corbett Rotor-Gene™ 6000 HRM real-time rotary analyzer. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA, 200 nM of each primer and 200 nM of each hydrolysis probe (ACTB - FAM™/BHQ^®-1, ERBB2 - CAL Fluor^® Gold 540/ BHQ^®-1, ERBB3 - CAL Fluor^® Orange 560/ BHQ^®-2, EGFR - CAL Fluor^® Red 610/ BHQ^®-2, ACTG1 - Quasar^® 705/ BHQ^®-2) using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95 ºC, 15 sec; 60 ºC, 15 sec.

Precise and highly reproducible discrimination

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Excellent reproducibility and discrimination across samples of low copy number with similar abundance levels was achieved. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (1.5-fold dilutions over a 89 - 450 copies per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ^®-1) hydrolysis probe using a fast cycling protocol (95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 20 sec) on the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer.

Broad linear dynamic range of up to 10 orders of magnitude using a fast cycling protocol

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Highly reproducible and 100% efficient qPCR was achieved across the 10 log-fold dilution series. Amplification plots were generated using 10-fold dilutions of a synthetic oligonucleotide target (60 – 6 x 106 copies per reaction) tested in triplicate using the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, synthetic DNA, 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ^®-1) hydrolysis probe using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95ºC, 3 sec; 60 ºC, 30 sec.

High performance with both standard and fast cycling protocols

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Excellent reproducibility and efficiency was achieved using both cycling protocols. Reactions were performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10-fold dilutions over a 0.1 - 100 ng per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using either a standard cycling protocol (95 ºC for 10 min followed by 40 cycles of 95ºC, 15 sec; 60 ºC, 60 sec) or a fast cycling protocol (95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 20 sec) on the Corbett Rotor-Gene ™ 6000 HRM real-time rotary analyzer.

Excellent benchtop stability

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KAPA PROBE FAST qPCR Master Mix offers the flexibility to perform experiments over multiple days. Reactions were left on the bench and in the dark for 0, 24, 48, 72, and 96 hours after assembly. Real-time PCR was performed in 20 μl volumes with KAPA PROBE FAST qPCR Master Mix, human genomic DNA (10-fold dilutions over a 0.1 - 100 ng per reaction range), 200 nM of each primer and 200 nM of hApoB100 (FAM/ BHQ®-1) hydrolysis probe using the following fast cycling protocol: 95 ºC for 3 min followed by 40 cycles of 95 ºC, 3 sec; 60 ºC, 30 sec. The data represents the average of 3 replicates for each DNA dilution and time point.

Licensed under U.S. Patent Nos. 5,338,671 and 5,587,287 and corresponding patents in other countries.

iCycler®, Prism®, Rotor-Gene™, BHQ®, Quasar®, CAL Fluor®, FAM™, and TaqMan® are trademarks or registered trademarks of their respective companies.