KAPA Blood PCR Kits
Product discontinued 1/1/2017
KAPA Blood DNA Polymerase is a second-generation enzyme derived through a process of directed evolution, and is the first DNA polymerase engineered specifically for the amplification of DNA directly from whole blood.
DNA fragments may be amplified directly from reactions containing 1%-20% (v/v) whole human blood without pretreatment of blood samples or DNA isolation, significantly reducing contamination risk, turnaround time, and the cost of genetic testing.
For Research Use Only. Not for use in diagnostic procedures.
Reduce contamination risk, turnaround time,
- Amplify DNA fragments directly from blood, minimizing the risk of sample cross-contamination
- Reduce the turnaround time and cost of genetic testing
Compatibility with existing workflows
- Integrate with existing protocols and detection methods using optimized, convenient 2X PCR Mixes:
– Blood PCR Mix A for fluorescent detection systems
– Blood PCR Mix B for endpoint PCR and detection of GC-rich targets
- Genetic testing
- Amplicons with a GC content <65% up to 3.5 kb in length; GC content >65% up to 1 kb
- Single amplicon or Multiplex PCR assays, using unlabeled or fluorescently-labeled primers
- Routinely-used detection methods
- Paternity testing using the PowerPlex® 16 System
Kits can be stored for up to 12 months at -20˚C.
Kits include KAPA Blood PCR Mix A or B (2X), which contain KAPA Blood DNA Polymerase, reaction buffer, dNTPs and MgCl2.
Starting Materialwhole blood
Input AmountEDTA blood (10% v/v) Disc punch from sample card or filter paper
KAPA Blood PCR Mix A
KAPA Blood PCR Mix B
- Genetic testing of humans, other mammals, and birds
- Amplicons with GC content <65% up to 3.5 kb in length, and amplicons with GC content >65% up to 1 kb
- Single amplicon or multiplex PCR assays, using unlabeled or fluorescently labeled primers
- Routinely used detection methods, including restriction endonuclease digestion, agarose gel electrophoresis/ethidium bromide staining, fluorescent capillary electrophoresis, DNA sequencing and dHPLC analysis
- Paternity testing using, for example, the PowerPlex® 16 System (Promega)
- Direct amplification from whole fresh or frozen blood, blood collected in EDTA tubes, or on FTA® Elute Cards, “Guthrie cards”, or filter paper.
- No need for DNA extraction in genetic testing protocols for humans, other mammals, and birds.
- Reduced contamination risk, turnaround time, and cost in genetic testing.
- Compatibility with existing workflows, standard protocols, and routinely used detection methods.
- Fresh or frozen whole blood
- Blood collected in EDTA (“lavender”) anticoagulant tubes (stored at 4°C for >2 years).
- Punches from Whatman 903® Specimen Collection Paper (“Guthrie cards”) on which blood has been collected/stored.
- Punches from regular filter paper on which blood has been collected/stored.
- Punches from Whatman FTA Elute Cards on which blood has been collected/stored (no elution step required).
- Punches from FTA Cards other than FTA Elute Cards used for blood collection/storage have to be treated according to the manufacturer’s recommendations prior to use in KAPA Blood PCR.
- Blood collected in heparin (green) anticoagulant tubes.
- KAPA Blood PCR Kits have also not been validated for the direct amplification of DNA from blood collected in citrate (light blue) or ACD (yellow) anticoagulant tubes.
- The blood sample used as template is too old, has not been stored properly, or is degraded.
- The blood sample used as template has been collected in heparin anticoagulant tubes.
- Primers are of a poor quality. Always dilute primers in 10 mM Tris-Cl, pH 8.5 and avoid using primers that have undergone multiple freeze-thaw cycles.
- The KAPA Blood PCR Mix has not been stored properly or has undergone too many freeze-thaw cycles. Always store KAPA Blood PCR Mix at -20°C or at 4°C for short-term use (≤1 week). If KAPA Blood PCR Mixes are used infrequently, it is recommended that smaller, single-use aliquots are prepared when kits are first received or thawed.
- The optimal concentration of blood included in a whole blood PCR using KAPA Blood PCR Mix A or B depends on the species, amplicon type, and application. Both KAPA Blood PCR Mixes have been validated with 1%–20% v/v whole human EDTA blood per reaction (i.e. 0.5–10 µL in a 50 µL reaction). For the best balance between sensitivity, specificity, and recovery of PCR product for downstream analysis, 5% – 10% v/v human EDTA blood per reaction is recommended as the starting point for most assays. For GC-rich, long or other difficult amplicons, optimal results may be obtained with more or less blood; the best concentration has to be determined empirically for such amplicons.
- Amplification from higher concentrations of blood (up to 40% v/v) has been achieved in selected cases.
- The optimal diameter of FTA Elute Card, “Guthrie card,” or filter paper punches/discs for KAPA Blood PCR depends on the reaction volume, amplicon type, and application.
- The optimal concentration of blood in KAPA Blood PCR for non-human species has to be determined empirically. Typically, the optimal concentration of mouse blood for most applications appears to be much lower (0.1%–5% v/v). Blood from bird species with nucleated erythrocytes typically has to be diluted to more closely approximate the DNA concentration (template copy number) present in the same volume of human blood. Dilutions should be prepared freshly just before PCRs are set up.
- The initial denaturation time is 5–10 minutes (94°C–95°C).
- The denaturation time in each cycle is at least 30 seconds (94°C–95°C).
- The annealing time in each cycle is at least 30 seconds (use the same annealing temperature as in the original protocol).
- The extension time in each cycle is at least 1 min/kb (72°C).
- Use the same number of cycles as in the original protocol. If a touchdown protocol is used in the existing assay, also use a touchdown protocol for the KAPA Blood PCR.
- A final extension step is strictly not required, but 1 min per kb at 72°C may be included.
- KAPA Blood PCR Kit A is recommended for assays employing highly sensitive fluorescent detection systems. It is the preferred KAPA Blood Kit for paternity testing using the PowerPlex16 System (Promega Corporation).
- KAPA Blood PCR Kit B has been formulated for optimal yields and is recommended for assays where amplified DNA is detected by agarose gel electrophoresis and ethidium bromide staining. It is the preferred KAPA Blood Kit for GC-rich and other difficult amplicons.
- Spin KAPA Blood PCR products for at least 5 minutes at maximum speed (14,000–17,000 x g) in a benchtop microfuge prior to post-PCR processing or analysis. If PCRs were performed in plates, centrifugation times may have to be increased significantly to compensate for the lower g-force limits of microplate centrifuges. To obtain the most compact pellet of organic debris (and facilitate recovery of the amplicon-containing supernatant), avoid handling of PCR products in a manner that may result in the distribution of debris across the inside surface of the tube or plate (e.g. do not invert tubes or plates).
- DNA sequencing and/or analysis by fluorescent capillary electrophoresis: carefully remove the cleared supernatant, process, and analyze using standard protocols for PCR products generated with isolated DNA as template. Purification of the cleared supernatant with a standard PCR cleanup kit prior to DNA sequencing is strongly recommended.
- RFLP analysis: Some restriction endonucleases (RE) will function fully in the cleared supernatants of KAPA Blood PCR products, whereas others will yield incomplete digests or will not digest at all. Whether or not a specific RE may be used in a direct post-PCR digest depends primarily on the activity requirements of the RE and has to be determined empirically. If incomplete digestion occurs, purification of the cleared supernatant with a standard PCR cleanup kit prior to RE digestion is recommended.