KAPA2G Fast Multiplex PCR Kits

High speed, high performance multiplex PCR without the need for optimization.

KAPA2G Fast Multiplex PCR Kits contain a second-generation (2G) enzyme derived through a process of molecular evolution. The polymerase was engineered for higher processivity and speed, offering significantly faster extension rates than wild-type Taq DNA polymerase. In addition to speed, KAPA2G Fast provides higher yields and sensitivity than competitor enzymes for highly multiplexed PCR.

KAPA2G Fast Multiplex Kits offer:

  • Higher yields and sensitivity
  • Uniform representation of all amplicons
  • Reduction in PCR cycling time up to 60%
  • High speed without compromising performance
  • Minimal optimization with master mix formulation
CodeDescriptionKit ContentsQtyUnit Price
KK5801 KAPA2G Fast Multiplex PCR Kit (100 x 25 µL rxn) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X master mix format optimized for multiplex PCR. Contains Mg2+ at a 1X conc. of 3 mM. **login for pricing
KK5802 KAPA2G Fast Multiplex PCR Kit (500 x 25 µL rxn) KAPA2G Fast HotStart DNA Polymerase in a convenient 2X master mix format optimized for multiplex PCR. Contains Mg2+ at a 1X conc. of 3 mM. **login for pricing

Product Description

KAPA2G Fast Multiplex PCR Kits are designed for Fast Multiplex PCR, offering faster cycling times and improved reaction efficiency compared to conventional Multiplex PCR assays performed with wild-type Taq DNA polymerase.

KAPA2G Fast Multiplex Mix (2X) is a ready-to-use cocktail containing all components for Fast Multiplex PCR, except primers and template. The Multiplex Mix contains KAPA2G Fast HotStart DNA Polymerase (1 U per 25 μl reaction), KAPA2G Buffer A (1.5X at 1X), dNTPs (0.2 mM each dNTP at 1X), MgCl2 (3.0 mM at 1X) and stabilizers.

KAPA2G Fast HotStart DNA Polymerase is an antibody-mediated hot start formulation of KAPA2G Fast DNA Polymerase, a second-generation enzyme engineered via molecular evolution. KAPA2G Fast DNA Polymerase was optimized for higher processivity and speed, offering significantly faster extension rates than wild-type Taq DNA polymerase. In the HotStart formulation, the enzyme is combined with a proprietary antibody which inactivates the enzyme until the first denaturation step. This eliminates spurious amplification products resulting from non-specific primer events during reaction setup and initiation, and improves overall reaction efficiency.

Multiplex PCR is a challenging application that typically requires more optimization than standard, single amplicon PCR assays. The key to successful Multiplex PCR is the ability to define a single set of reaction parameters (reagent concentrations and cycling parameters) that allows for all primers to anneal with high specificity to their target sequences and be extended with the same efficiency. Primer design, as well as the enzyme and buffer system, are critical factors in this challenge.

The KAPA2G Fast Multiplex PCR Kit offers faster and more efficient Multiplex PCR than competitor enzymes based on wild-type Taq DNA polymerase. The increased speed and processivity of the engineered KAPA2G Fast HotStart DNA Polymerase results in a significant decrease in cycling times, whilst still maintaining the ability to efficiently amplify difficult fragments. The uniquely formulated KAPA2G Fast Multiplex Mix facilitates primer annealing and highly specific amplification of a wide range of amplicon sizes and GC contents, resulting in more even amplification of all target fragments.

Product Applications

The KAPA2G Fast Multiplex PCR Kit is ideally suited for end-point, Fast Multiplex PCR of multiple DNA fragments, ranging in size from 50 – 1,500 bp. Up to 30 different primer pairs may be combined into a single assay using the protocols provided in this Technical Data Sheet.

The kit is ideally suited for:

  • Typing of transgenic organisms
  • Amplification of microsatellites
  • Typing and detection of pathogens
  • Amplification of multiple DNA fragments for SNP genoptyping
 

High performance multiplex PCR.

KAPA2G Fast Multiplex PCR Kits are based on a second- generation (2G) polymerase capable of synthesizing DNA faster than wild-type Taq and other DNA polymerases. A total extension time of 15 sec/cycle is sufficient for many multiplex assays, compared to 60 - 90 sec/cycle for competitor kits containing wild-type Taq.

Multiplex PCR (4- and 8-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I. Reactions (25 μl) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 ng - 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

60% reduction in total cycling time.

Fast PCR protocols using KAPA2G Fast Multiplex PCR Kits are based on reduced extension times that allow for up to 60% reduction in PCR cycling time, without the risk of compromising reaction performance, or having to invest in specialized PCR consumables or instrumentation.

Fast Multiplex PCR with KAPA2G Fast Multiplex PCR Kits. Multiplex PCR with wild-type Taq typically requires very long annealing and extension times to allow primer annealing and extension of all primers in the multiplex. KAPA2G Fast Multiplex PCR Kits contain KAPA2G Fast HotStart DNA Polymerase, a second-generation (2G) polymerase engineered via molecular evolution to synthesize DNA at a faster rate. Total PCR cycling times required for 4-plex, 8-plex and 12-plex multiplex PCRs (30 cycles, set up according to the manufacturers’ recommendations) with KAPA2G Fast Multiplex PCR Kits and Competitor Q Multiplex PCR Kit (which contains wild- type Taq DNA polymerase) are shown. Time savings of 40 - 60% are possible with the KAPA2G Fast Multiplex PCR Kit.

Uniform representation of all amplicons in 12-plex PCR.

12-plex Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I. Achieving uniform representation of all amplicons in a complex multiplex assay is a challenge due to amplification bias, a result of differences in amplicon length, secondary structure and priming efficiency. Reactions (25 μl) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 ng - 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Successful multiplex PCR with difficult, GC-rich targets.

6-plex GC-rich Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I. Successful Multiplex PCR with wild-type Taq is limited to easy, simple targets that can be amplified with equal efficiency. The improved processivity of the engineered KAPA2G Fast DNA Polymerase allows uniform multiplex PCR of a broad range of difficult targets. Reactions (25 μl) contained 1X PCR Master Mix (KAPA and Competitor Q) or 1X PCR Buffer, 3 mM MgCl2, 0.2 mM of each dNTP and 1 U of hot start Taq DNA Polymerase (home brew multiplex reagents, with Competitor I). Human genomic DNA was used as template (250 ng - 2 ng per reaction), and primers were supplied at 0.2 μM each. DMSO (5%) and KAPA Enhancer 1 (1X) was added to all reactions. Cycling was performed according to manufacturers’ recommendations (30 cycles). Amplicons range in size from 241 - 642 bp, and in GC content from 72.7 - 83.8%.