KAPA3G Plant PCR Kits

Evolved to solve. Direct PCR from plant tissue.

The KAPA3G Plant PCR Kit is based on a novel, third-generation (3G) DNA polymerase, engineered via molecular evolution for improved tolerance to common plant-derived PCR inhibitors such as polyphenolics and polysaccharides. Kits are optimized for fast and efficient amplification of plant DNA from crude samples, DNA containing carry-over inhibitors from crude extraction methods, and purified DNA.

Key features of the KAPA3G Plant PCR Kit include:

•Fast PCR direct from plant tissues such as leaf discs, seeds and crude plant extracts. 

•Streamlined workflows for transgenic screening. 

•Improved PCR success rates and reproducibility. 

•Efficient amplification of long and difficult targets from all sample types.

Product Description

Amplification of plant-derived DNA is a challenging application due to the diversity of plant tissue types and the potent PCR inhibitors contained within the tissue. The KAPA3G Plant PCR Kit is optimized for the successful amplification of DNA from crude plant samples, DNA containing carry-over inhibitors from crude extraction methods, as well as purified DNA.

The KAPA3G Plant PCR Kit contains a novel DNA polymerase, engineered via a process of molecular evolution, for improved tolerance to common plant-derived PCR inhibitors such as polyphenolics and polysaccharides. The unique characteristics of the enzyme result in robust amplification across a wide range of plant sample types, amplicon lengths, and crude extraction methods. The kit contains 4 separate components: 1) KAPA Plant PCR Buffer (2X) is a ready-to-use cocktail containing all components except DNA polymerase, primers, and template. This 2X buffer contains 3 mM MgCl2; 2) KAPA3G Plant DNA Polymerase is a blend of an engineered Taq-based (Type A) DNA polymerase and a modified archaeal (Type B) DNA polymerase. This enzyme blend is combined with proprietary antibodies that inactivate the enzymes prior to the first denaturation step; 3) KAPA Plant PCR Enhancer is supplied as an optional additive to improve PCR performance for difficult samples and assays where MgCl2 titration fails to improve results; 4) Additional MgCl2 (25 mM) is supplied for assays that require MgCl2 optimization.

DNA fragments generated with the KAPA3G Plant PCR Kits are A-tailed and suitable for use with TA cloning vectors.

Product Applications

The KAPA3G Plant PCR Kit is designed for the amplification of DNA fragments ≤5 kb in length from a range of plant samples including:

  • Crude extractions of plant DNA containing carry-over inhibitors
  • Directly from leaf discs, seed samples, and other plant tissue samples
  • Samples containing significant concentrations of plant-derived compounds
 

KAPA3G Plant PCR Kits contain a novel DNA polymerase engineered via a process of molecular evolution for improve tolerance to common plant-derived inhibitors.

Direct PCR from a variety of plant species and tissue types

The KAPA3G Plant PCR Kit is capable of amplifying DNA fragments from a variety of templates, including purified DNA (+), leaf discs (L) or seeds (S). Plant genomic DNA was purified from all species using a commercial DNA purification kit. A Harris Uni-CoreTM sampling tool (0.5 mm diameter) was used to sample leaves (all species) or seeds (all species except tobacco and Arabidopsis; for these one crushed seed was used per reaction). PCRs (50 μL) contained the crude sample or 1-10 ng purified DNA (depending on the species), and 40 cycles were performed in all cases. Targets ranged between 500 and 900 bp, and reaction products were analyzed in a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Streamline workflows and improve the reproducibility of results

Direct PCR using the KAPA3G Plant PCR Kit outperforms CTAB extraction and standard PCR using wild-type Taq, in significantly shorter turnaround times. CTAB-extracted DNA or leaf discs were used as templates for the amplification of targets from maize (860 bp) and tobacco (735 bp), using the KAPA3G Plant PCR Kit (top panel) or wild-type Taq (bottom panel). For each species, genomic DNA was purified in triplicate using a common CTAB extraction method. Crude material was sampled in triplicate using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. CTAB-extracted DNA and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Direct PCR with the KAPA3G Plant PCR Kit was completed in 45 min. In contrast, the CTAB extraction protocol required ~2 h, and the amplification with wild-type Taq 1.5 h to complete. The KAPA3G Plant PCR Kit outperformed wild-type Taq when using both CTAB- extracted DNA and crude sample. Reaction products were analyzed in a 1% agarose gel. KAPA Express Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Evolved DNA polymerase enables higher performance PCR direct from plant tissue

The KAPA3G Plant PCR Kit outperforms competitor kits across a broad range of plant species, with both crude samples and purified DNA. For each species, genomic DNA was purified using a commercial DNA purification kit. Crude leaf material was sampled using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. Purified DNA (1-10ng per reaction, depending on species) and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Reaction setup and cycling were performed according to each manufacturers’ recommended protocol. Targets ranged between 500 bp and 900 bp and reaction products were analyzed in a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

Successful amplification of long targets from crude samples

The KAPA3G Plant PCR Kit is capable of amplifying long targets from crude samples and purified DNA with equal efficiency. Targets of different lengths (297 bp and 4100 bp from tobacco, 640 bp from tomato, 1221 bp from grapevine, and 1448 and 2249 from potato) were amplified from purified DNA (+) or leaf discs (L) using the KAPA3G Plant PCR Kit. For each species, genomic DNA was purified using a commercial DNA purification kit. Crude material was sampled using a 0.5 mm diameter Harris Uni-CoreTM sampling tool. Purified DNA (1-10 ng per reaction, depending on species) and crude samples were used as templates in 50 μL PCRs, with 40 cycles of amplification. Reaction products were analyzed on a 1% agarose gel. KAPA Express DNA Ladder (100, 200, 400, 800, 1600, 4000, 8000 bp) was used as a MW marker.

License Information

Licensed under U.S. Patent nos. 5,338,671, 5,587,287, 5,436,146 (claims 6-16) and corresponding patents in other countries.