The KAPA Library Amplification Primer Mix (10X) contains primers that target the P5 and P7 regions of Illumina TruSeq® and dual-indexed adapters. The primer mix is formulated to limit primer depletion and over-amplification.
High-fidelity PCR is used to selectively enrich library fragments carrying appropriate adapter sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment of libraries, minimizing amplification bias is critical to ensure uniform sequence coverage. 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’s 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.
- Real-time amplification workflows are amenable to automation.
- Real-time amplification plots provide quality metrics for individual enriched libraries, eliminating expensive and time-consuming post-enrichment gel electrophoresis and identifying inconsistencies in library preparation.
- Seamless integration with KAPA Library Quantification Kits.
The kit contains KAPA HiFi HotStart Real-time PCR Master Mix (2X), a ready-to-use cocktail containing all components for PCR, except primers and templates.
On the other hand, The 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.
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.
KAPA HiFi HotStart DNA Polymerase has 5’g3′ polymerase and 3’g5′ exonuclease (proofreading) activities, but no 5’g3′ exonuclease activity. The strong 3’g5′ exonuclease activity results in superior accuracy during DNA amplification. The error rate of KAPA HiFi HotStart DNA Polymerase is ~2.8 x 10-7. This fidelity is approximately 100X higher than that of wild-type Taq and up to 30X higher than polymerase blends. The presence of SYBR Green I dye in the reaction does not compromise fidelity. DNA fragments generated with KAPA HiFi HotStart Real-time PCR Master Mix (2X) may be used for routine downstream applications, including restriction enzyme digestion and sequencing. PCR products generated with KAPA HiFi HotStart Real-time PCR Master Mix (2X) are blunt-ended, but may be 3′-dA-tailed for cloning into TA cloning vectors.
