KAPA Blood PCR Kits

Evolved for whole blood PCR.

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.

Product Highlights

Amplification of a 459 bp fragment of exon 19 of the human Duchenne muscular dystrophy gene. Reactions (50 µL) contained different amounts of blood (0-20% v/v, as indicated) from EDTA anticoagulant tubes stored at 4°C (lanes 1-6), or punches from an FTA® Elute Card (lane 7) or “Guthrie” card (lane 8). Control reactions, performed with 10 ng or 1 ng purified human genomic DNA and KAPA Blood PCR Mix B (lanes 9-10), or wild-type Taq Polymerase (lanes 11 and 12) are indicated on the right. A standard 3-step cycling profile (35 cycles) with an initial denturation of 5 minutes (95°C) and 1 minute extension time per cycle was used in all reactions. Data on file.

Reduce contamination risk, turnaround time,
and cost

  • 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

High-throughput genotyping of mice used in investigations into the relationship between metallothionein (MT) expression and mitochondrial function. To distinguish mice carrying the knockout mutation on one or both alleles from homozygous wild-type mice, a fragment of the MT gene was amplified directly from mouse blood collected on “Guthrie” cards, using KAPA Blood PCR Kit B. A standard 3-step cycling protocol (30 cycles) with an initial denaturation of 10 min (95°C) was employed. PCR products were cleared by centrifugation, electrophoresed in a 2.5% agarose gel, and detected by ethidium bromide staining. A single larger band corresponds to a homozygous knockout mouse (KO), whereas a single smaller band corresponds to the homozygous wild-type (WT). Heterozygotes (Het) yield a double band representative of both the KO and WT alleles. Image courtesy of North-West University. Data on file.

Direct amplification of non-human blood

  • Direct amplification of DNA from the blood of non-human species, such as other mammals and birds
  • Reduces cost and turnaround time in PCR-based veterinary testing
Applications
  • 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

 

Kit Specifications and Contents / Storage

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.

Specifications

Spec
Description
Starting Material
whole blood
Input Amount
EDTA blood (10% v/v) Disc punch from sample card or filter paper

Contents

Component
KAPA Blood PCR Mix A
KAPA Blood PCR Mix B
FAQs
What are the major benefits of using the KAPA Blood PCR Kit?

  • 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.

What is the enzyme in KAPA Blood PCR Kits?
KAPA Blood PCR Kits contain KAPA Blood DNA Polymerase, a second-generation DNA polymerase and the first to be engineered specifically for the amplification of DNA directly from whole blood. KAPA Blood DNA Polymerase was engineered through high-throughput directed evolution to be resistant to common inhibitors present in blood. This novel enzyme allows for consistent and reliable amplification of DNA directly from blood, with results comparable to the amplification from isolated DNA using wild-type DNA polymerases.
Which type of blood samples may be used as templates in whole blood PCR using KAPA Blood PCR Kits?

  • 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.

Which type of blood samples are unsuitable as templates in whole blood PCR using KAPA Blood PCR Kits?

  • 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.

What are the most common causes of poor results (e.g., failed amplification, low yields, and/or non-specific amplification)?

  • 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.

How much blood should be used in a KAPA Blood PCR?

  • 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.

Does whole blood PCR with KAPA Blood PCR Kits require any pretreatment of blood?
No, whole fresh or frozen blood, EDTA blood, FTA Elute Card punches, “Guthrie card” punches, or filter paper punches may be added directly to reactions containing the KAPA Blood PCR Mix, primers, and PCR-grade water. No pre-PCR lysis, heating, and/or centrifugation of blood samples are required. For amplification from blood stored on FTA Cards other than FTA Elute Cards, card punches have to be treated according to the manufacturer’s recommendations prior to being used as templates in KAPA Blood PCR.
Does whole blood PCR with KAPA Blood PCR Kits require modified cycling parameters?
In most cases, whole blood PCRs with KAPA Blood PCR Kits can be performed with exactly the same cycling parameters as used in the original protocol with isolated DNA as template. However, please ensure that:

  • 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.

Can KAPA Blood PCR Kits be used for the amplification of long and/or difficult amplicons directly from blood?
DNA fragments up to 3.5 kb (with a GC content <65%) have been amplified successfully directly from blood using KAPA Blood PCR Kits. Yields of long amplicons may be improved by including Tween 20® in the reaction to a final concentration of 0.1% v/v. Direct amplification of GC-rich amplicons (GC content <65%) is possible in the presence of DMSO, but the maximum amplicon length is limited to ≤1 kb. For GC-rich amplicons, include DMSO to a final concentration of 5% v/v in the reaction. Amplification of certain “difficult” amplicons with a GC content <65%, such as those with stable secondary structure, may also be improved by including DMSO in the reaction.
Can KAPA Blood PCR Kits be used for pathogen detection directly in blood?
DNA fragments derived from pathogens have been amplified successfully from whole blood using KAPA Blood PCR Kits. However, these kits have not been validated for routine pathogen detection. Amplification of pathogen amplicons directly from whole blood is likely to be less sensitive than amplification from isolated DNA.
Can KAPA Blood PCR Kits be used for DNA amplification directly from the blood of non-human species?
Yes, direct amplification of DNA fragments from blood of other mammals (e.g., mice and cats) and several bird species, collected in a suitable manner (see Question 7) has been achieved.
Can KAPA Blood PCR Kits be used for the amplification of DNA directly from other crude samples?
KAPA Blood DNA Polymerase was evolved specifically for DNA amplification directly from human EDTA blood, and KAPA Blood PCR Mixes A and B have been formulated optimally for this template. However, the inhibitor resistance of the polymerase makes it suitable for direct amplification from other types of crude samples. Positive results with buccal swabs and amniotic fluid have been reported. Optimal reaction conditions for non-blood templates have to be determined empirically. For amplification from crude samples for which KAPA Blood PCR Kits do not yield satisfactory results, KAPA2G Robust HotStart PCR Kits are recommended.
Can KAPA Blood PCRs be set up at room temperature?
Reaction setup at room temperature is possible due to the intrinsic hot-start nature of whole blood PCR – template DNA is only released when hematocytes are lysed during the initial denaturation step. However, the KAPA Blood DNA Polymerase is active at room temperature and it is recommended that primers be designed carefully to eliminate primer-dimer formation at room temperature.
What are the storage recommendations for KAPA Blood PCR Kits?
Always store KAPA Blood PCR Mixes 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. When stored under these conditions and handled correctly, all kit components will retain full activity for 6 months from date of receipt.
What is the difference between KAPA Blood PCR Kits A and B, and which should I use for my assay?
KAPA Blood PCR Kits A and B contain KAPA Blood DNA Polymerase in two optimized, convenient 2X ready-mix formulations, containing all components required for whole blood PCR, except primers and template (blood). The enzyme has been formulated in different buffers for different applications:

  • 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.

Does whole blood PCR require special post-PCR processing prior to analysis of reaction products?
Using whole blood instead of isolated DNA as the template in a PCR may have implications for downstream processing and analysis, due to the presence in the reaction product of denatured protein and other organic debris released from hematocytes. A significant fraction of these compounds may be eliminated by centrifugation of whole blood PCR products. However, cleared supernatants may still contain salts and other compounds that are inhibitory to some downstream processes and detection methods. For optimal results, the following are recommended:

  • 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.

Can PCR products generated with the KAPA Blood PCR Kit be digested, cloned, and sequenced?
Yes, PCR products generated with the KAPA Blood PCR Kit have the same characteristics as PCR products generated with wild-type Taq polymerase. They may be sequenced or digested with restriction endonucleases using standard protocols. Products are 3′-dA-tailed and may be used for TA cloning, or may be blunt-ended or digested with restriction enzymes prior to cloning. For best results, purification of PCR products using any standard PCR cleanup kit is recommended.
Can PCR products generated with the KAPA Blood PCR Kit be analyzed by dHPLC?
Yes, KAPA Blood PCR Mixes have been formulated not to contain any compounds that are refractory to analysis of PCR products by dHPLC; however, the organic debris present in whole blood PCR products (particularly denatured protein) may adversely affect dHPLC systems. Purification of KAPA Blood PCR products with any standard PCR cleanup kit is therefore recommended prior to dHPLC analysis.

Ordering

Kits include KAPA Blood PCR Mix A or B (2X), which contain KAPA Blood DNA Polymerase, reaction buffer, dNTPs and MgCl2.

Kit Code
Roche Cat. No
Description
Kit Size
How to buy
KK7004
07961570001
Mix A, 1000 x 25 µL reactions
12.5 mL
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