Rapid extraction and amplification of DNA from mouse tissue in less than 1 hour.
KAPA Mouse Genotyping Kits contain:
- KAPA Express Extract - 15 minute extraction using no organic solvents and requiring no pH neutralization or centrifugation.
- KAPA2G Fast Genotyping Mix - 45 minute amplification using a novel DNA polymerase engineered for high processivity and extreme speed.
- Available in a convenient master mix format with loading dye, in both hot start and non-hot start formulations.
KAPA Mouse Genotyping Kits include KAPA Express Extract, a novel thermostable protease and buffer system that allows the extraction of PCR-ready DNA from mouse tissue in as little as 15 minutes, and KAPA2G Fast Genotyping Mix with dye, containing a DNA polymerase engineered via a process of molecular evolution for high processivity and extreme speed. The combination of KAPA Express Extract and KAPA2G Fast Genotyping Mix allows for the reliable extraction and amplification of DNA fragments from mouse tissue in 1 hour, as compared to ≥1 day with conventional protocols.
The KAPA Express Extract system has been designed for optimal tissue lysis and sample preservation. Unlike existing protocols that rely on proteinase K digestion, extractions using KAPA Express Extract are conveniently performed in a single-tube, without the need for hazardous chemicals and multiple washing steps.
Extracted DNA is then amplified with KAPA2G Fast Genotyping Mix (2X) with dye, a ready-to-use master mix containing all components for Fast PCR, except primers and template. The master mix is designed for routine Fast PCR and improved performance, compared to wild-type Taq DNA polymerase. The master mix with dye additionally contains two inert dyes, which allow for the analysis of reaction products by gel electrophoresis directly after completion of the PCR, i.e. without the need to add a DNA loading solution.
Current workflows for the extraction and amplification of DNA for mouse genotyping can benefit from improvements in throughput, turnaround time and reliability.
Template DNA for mouse genotyping is routinely prepared from ear or tail biopsy samples. Typically, samples are incubated for several hours or overnight with Proteinase K, followed by purification of the DNA to remove salts and detergents. Alternatively, DNA is released from the mouse tissue by heating the sample in an alkaline solution, followed by neutralization in a Tris-HCl buffer with an acidic pH. Proteinase K protocols yield extracts with a higher DNA concentration and quality, but are laborious and time-consuming. Heat lysis protocols allow for rapid generation of PCR-ready DNA, but samples vary in quality and the concentration of amplifiable template, leading to reduced PCR success rates with wild-type DNA polymerases.
Kapa Biosystems offers a unique solution for mouse genotyping. KAPA Mouse Genotyping Kits KAPA Express Extract DNA Extraction Kits contain a novel thermostable protease and buffer system designed for rapid and efficient, single-tube mouse tissue lysis. Good quality, PCR-ready DNA can be generated in as little as 15 min with minimal handling, thereby reducing the risk of sample loss or contamination. The process yields sufficient template for multiple assays and is easily scaled to handle samples in a 96-well format. KAPA2G Fast Genotyping Mix with dye is ideally suited for the fast and reliable amplification of DNA fragments across a wide range of amplicon lengths and GC contents. This ready-to-use cocktail contains KAPA2G Fast DNA polymerase, a novel enzyme engineered for improved processivity and speed. Together, these reagents allow for significantly reduced turnaround times and improved success rates in mouse genotyping.
The KAPA Mouse Genotyping Kit outperforms crude DNA extraction methods used for mouse genotyping, in significantly shorter turnaround times.
Results for five amplicons (312 – 915 bp) generated with the KAPA Mouse Genotyping Kit (1) were compared to those obtained with two commonly used methods (2 and 3). With the KAPA Mouse Genotyping Kit, DNA lysates were prepared from mouse tails with the rapid (15 min), single-tube KAPA Express Extract system. Amplification with the KAPA2G Fast HotStart Genotyping Mix with dye was completed in 45 min. In contrast, DNA lysates were prepared with a ~3.5-hour Proteinase K protocol (2) or a rapid (~2.5 h) alkaline lysis method (3) (top panel). In both cases, amplification was performed with wild-type Taq (1.5 h cycling protocol). Results obtained with the KAPA Mouse Genotyping Kit were equal or better (more specific) than those obtained with other methods, which (depending on the exact DNA extraction protocol used), may take at least four times as long, or up to 1 day to complete.
The total time required for DNA extraction and amplification were compared using the KAPA Mouse Genotyping Kit, Proteinase K and wild-type Taq, and alkaline lysis and wild-type Taq.
The KAPA Mouse Genotyping Kit performs as well or better than other commercial kits used for mouse genotyping at lower cost.
The KAPA Mouse Genotyping Kit performs as well or better than other commercial kits used for mouse genotyping. Results for five amplicons (312 – 915 bp) generated with the KAPA Mouse Genotyping Kit were compared to those obtained with the Phire® Animal Tissue Direct PCR Kit (Finnzymes), designed for direct PCR from animal tissue without purification, and the REDExtract-N-Amp™ Tissue PCR Kit (Sigma), designed for rapid extraction and amplification of genomic DNA from animal tissues. All kits were used according to manufacturer’s recommendations. DNA extraction and amplification was completed in 1 hour with the KAPA kit. The Finnzymes kit contains an engineered enzyme suitable for fast cycling. The protocol was completed in 37 min (as no DNA extraction is required), but produced non-specific amplicons and a low yield of the amplicon with the highest GC content (64 % GC). The Sigma kit employs a rapid (15 min) DNA extraction protocol, but DNA amplification (with wild-type enzyme) required 1.5 h cycling time.
Cost per reaction for KAPA Mouse Genotyping Kit, Phire® Animal Tissue Direct PCR Kit (Finnzymes), and REDExtract-N-Amp™ Tissue PCR Kit