KAPA2G Fast Multiplex Kits

Overview

KAPA2G Fast Multiplex PCR Kits contain a second-generation (2G) enzyme derived through our directed evolution technology. KAPA2G FAST HotStart DNA Polymerase is an antibody-mediated hot start formulation engineered for higher processivity and speed, offering significantly faster extension rates than wild-type Taq DNA polymerase. In addition to speed, KAPA2G Fast Multiplex Kit provides higher yields and sensitivity than competitor enzymes, allowing for more uniform multiplexed PCR.*

Product highlights

Improved sensitivity, specificity, and yields compared to competitor kits

Uniform representation of all amplicons
Successful multiplex PCR with difficult, GC-rich targets

View data showing superior performance in terms of speed, sensitivity and uniform representation of all amplicons in multiplex PCR using KAPA2G Fast Multiplex PCR Kits compared to competitor kits.

Figure 1. Multiplex PCR (8-plex) performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I Kits. 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 – 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Figure 2. 12-plex Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I Kits. 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 - 2 ng per reaction), and primers were supplied at 0.2 μM each. Cycling was performed according to manufacturers’ recommendations (30 cycles).

Increased speed without compromising performance

60% reduction in cycling time
Extension times as low as 15 seconds
Successful multiplex PCR with difficult targets

View data showing faster reaction times in multiplex PCR reactions using KAPA2G Fast Multiplex PCR Kits compared to competitor kits.

View data showing better multiplex PCR amplification compared to competitor kits of samples with high GC-rich regions using KAPA2G Fast Multiplex PCR Kits.

Figure 3. 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 for primer annealing and extension of all primers in the multiplex. 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.

Figure 4. 6-plex GC-rich Multiplex PCR performed with the KAPA2G Fast Multiplex PCR Kit, Competitor Q and Competitor I Kits. 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 for 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) were 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%.