KAPA Real-Time Library Amplification Kits

A novel library amplification solution for optimizing PCR cycle number and reducing PCR-induced bias.

High fidelity PCR is used to selectively enrich library fragments carrying appropriate adaptor sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment of libraries, minimizing amplification bias is critical to ensure uniform coverage. Introduction of amplification bias requires more total sequencing in order to obtain sufficient coverage in regions of the genome that are underrepresented, increasing sequencing costs. Amplification bias occurs when a DNA polymerase is unable to amplify all targets within a complex population of library DNA with equal efficiency. Bias is further exacerbated when libraries are over-amplified.

KAPA Real-Time PCR Library Amplification Kits are designed to address both sources of PCR-induced bias. The novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity, is capable of balanced amplification of complex library DNA. Real-time monitoring of library amplification provides additional information required to minimize over-amplification. Benefits of performing high fidelity, real-time PCR for next-generation sequencing library amplification include:

  •  Real-time monitoring of amplification allows precise control over the optimal number of PCR cycles, reducing the uncertainty of when to terminate enrichment PCR. 
  •  Real-time amplification workflows are amenable to automation because post-PCR DNA electrophoresis is not required.
  •  Real-time amplification plots provide quality metrics for individual enriched libraries, eliminating expensive and time-consuming post-enrichment gel electrophoresis.
  •  Real-time library amplification integrates seamlessly with qPCR-based KAPA Library Quantification Kits.
CodeDescriptionKit ContentsQtyUnit Price
KK2701 KAPA Real-Time Library Amplification Kit (50 x 50 µL reactions) 50 x 50 µL reactions. Illumina series. Each kit contains 1 x 1.25 ml KAPA HiFi HotStart Real-Time PCR Master Mix (2X) and 4 x 1.5 ml Fluorescent Standards. **login for pricing
KK2702 KAPA Real-Time Library Amplification Kit (250 x 50 µL reactions) 250 x 50 µL reactions. Illumina series. Each kit contains 1 x 6.25 ml KAPA HiFi HotStart Real-Time PCR Master Mix (2X) and 4 x 1.5 ml Fluorescent Standards. **login for pricing

KAPA Real-Time Library Amplification Kits contain KAPA HiFi HotStart Real-Time PCR Master Mix (2X), a ready-to-use cocktail containing all components for PCR, except primers and template. The 2X Master Mix contains KAPA HiFi HotStart DNA Polymerase in a proprietary reaction buffer, dNTPs, MgCl2 (2.5 mM at 1X), SYBR® Green I dye and stabilizers. Four fluorescent standards are supplied, and are used to define a window for optimal amplification.

#mce_temp_url#KAPA HiFi HotStart DNA Polymerase is an antibody-based hot start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA polymerase exhibiting industry-leading performance in comparison with other high-fidelity (B-family) DNA polymerases and polymerase blends. KAPA HiFi DNA Polymerase was engineered for increased affinity to DNA, without the need for accessory protein domains. The intrinsic high processivity of the enzyme results in significant improvements in yield, sensitivity, speed, target length and the ability to amplify difficult amplicons. These enhancements result in lower amplification bias which leads to more uniform sequence coverage. In the HotStart formulation, a proprietary antibody inactivates the polymerase 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.

Real-time, high fidelity amplification of next-generation DNA sequencing libraries

Enzyme bias – along with other artifacts such as PCR-induced errors, adaptor dimers, PCR duplicates, and chimeras – is exacerbated by over-amplification, while under-amplification results in insufficient yields. Inherent uncertainty in the outcome of end-point PCR often demands downstream validation of library quality by electrophoresis. KAPA Real-Time PCR Library Amplification Kits provide a high fidelity, real-time PCR method for rapid and convenient amplification of libraries. Precise control over the number of cycles required for optimal amplification eliminates expensive and time-consuming post- enrichment gel electrophoresis.

Libraries are amplified using a SYBR® Green I-based real-time, high fidelity PCR master mix (left panel). Four triplicate wells of the PCR plate contain fluorescent reference standards representing a range of distinct DNA concentrations. Reactions terminated between standards 1 and 3 represent the optimal library amplification range (grey box), depicted here from cycle 10- 14. Gel image illustrating the enrichment of a typical library stopped at different amplifica- tion cycles relative to the fluorescent standards supplied in the KAPA Real-Time Library Amplification Kit (right panel). Low and high molecular weight artifacts increase progressively with additional cycles.
Superimposed amplification plots for reactions terminated at the lower bound (hashed line, cycle 10) or upper bound (solid line, cycle 14) of the targeted concentration range (grey box). Library amplification reactions should ideally be terminated anywhere within the indicated target concentration range.
Example of real-time, high fidelity amplification of multiple libraries. 20 libraries, spanning a ~64-fold concentration range (6 cycles), were simultaneously amplified and terminated after 14 cycles. 14 of the 20 libraries fall within the targeted amplification range. The remaining 6 libraries could either be used as is, noting that they may be outside the optimal concentration range, or they could be re-amplified individually or in high- or low-concentration groups.

KAPA Real-Time PCR Library Amplification Kit workflow 

License Information

KAPA Real-Time PCR Library Amplification Kits are developed, designed and sold exclusively for research purposes and in vitro use. Neither the product, nor any individual component, has been tested for use in diagnostics or for drug development, nor is it suitable for administration to humans or animals. Please refer to the MSDS, which is available on request.

Certain applications of this product are covered by patents issued to parties other than Kapa Biosystems and applicable in certain countries. Purchase of this product does not include a license to perform any such applications. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which the product is used.

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

Certain applications of this product are covered by patents issued to parties other than Kapa Biosystems and applicable in certain countries. Purchase of this product does not include a license to perform any such applications. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which the product is used.

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056, 6,171,785, and 5,928,907 (claim numbers 12-24, 27-28). The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

This product is provided under an agreement between Molecular Probes, Inc. and Kapa Biosystems Inc., and the manufacture, use, sale or import of this product is subject to one or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Molecular Probes, Inc. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851.

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

The purchase of this product includes a limited, non-transferable license under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein.

SYBR® is a registered trademark of Molecular Probes, Inc, Oregon.