KAPA Blood PCR Kits featured in "Direct PCR from Whole Blood" poster from ARUP Institute for Clinical and Experimental Pathology and the Department of Pathology at the University of Utah. Download here >>

Product Description

KAPA Blood PCR Kits are available in two optimized, easy-to-use 2x formats containing all PCR components except primers and template (whole blood).

KAPA Blood PCR Mix A is preferred for assays employing highly sensitive fluorescent detection systems. KAPA Blood Mix B is formulated for higher yields and GC-rich amplicons and is recommended when analysis is by agarose gel electrophoresis and ethidium bromide staining. Products generated with KAPA Blood PCR Mixes A or B may be analyzed by restriction endonuclease digestion, DNA sequencing or dHPLC analysis.

Product Applications

KAPA Blood PCR Kits have been validated for the direct amplification of DNA fragments from fresh or frozen whole blood, blood collected in EDTA anti-coagulant tubes, on FTA® Elute Cards, Whatman 903® Specimen Collection Paper (“Guthrie cards”) or regular filter paper. The optimal amount of blood per reaction depends on the species, amplicon type and application. KAPA Blood PCR Kits are recommended for the following:

• Genetic testing of humans, other mammals and birds.
• Amplicons with a 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 unlabelled or fluorescently labelled primers.
• Routinely used detection methods including restriction endonuclease digestion, agarose gel electrophoresis/ethidium bromide staining, fluorescent capillary electrophoresis, DNA sequencing and dHPLC analysis.
• KAPA Blood PCR Kits are ideally suited for paternity testing using the PowerPlex® 16 System (Promega).

Reduce contamination risk, turnaround time and cost of genetic testing

High-throughput genetic testing directly from blood has not been feasible with wild-type polymerases, due to the presence of multiple PCR inhibitors in whole blood. KAPA Blood DNA Polymerase was evolved in this environment and offers the ability to amplify DNA fragments directly from blood, without any pretreatment of blood samples or DNA isolation. This not only reduces the cost and turnaround time of genetic testing, but also significantly reduces the risk of sample cross-contamination associated with the need for DNA extraction.

Amplification of a 459 bp fragment of exon 19 of the human Duchenne muscular dystrophy gene directly from whole human blood, using KAPA Blood PCR Mix B.

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 a “Guthrie card” (lane 8) as template. Control reactions, performed with 10 ng or 1 ng purified human genomic DNA as template and KAPA Blood PCR Mix B (lanes 9 and 10), or wild-type Taq polymerase (lanes 11 and 12) are included on the right. A standard 3-step cycling profile (35 cycles) with an initial denaturation of 5 min (95°C) and 1 min extension time per cycle was used in all reactions.

Compatibility with existing workflows and detection methods

KAPA Blood DNA Polymerase is supplied in two optimized, convenient 2x PCR Mixes, which are easily integrated with existing protocols and detection methods. Reaction products are centrifuged to recover amplified DNA from hematocyte debris.

Paternity testing using fluorescent capillary electrophoresis

Typical result obtained in a PowerPlex16® (Promega Corporation) paternity test using KAPA Blood PCR Mix A, with blood collected on Whatman 903® Specimen Collection Paper (“Guthrie cards”) as template. The test is based on the amplification of 16 loci (15 STR loci and amelogenin) in a single Multiplex PCR using primers labelled with three different fluorophores. Reactions were set up and performed according to manufacturers’ recommendations. Reaction products were centrifuged and cleared supernatants were analyzed directly by fluorescent capillary electrophoresis using standard protocols. Image courtesy of Unistel Medical Laboratories.

Genetic testing based on RE digestion of DNA fragments amplified directly from whole blood

Incorporation of whole blood PCR in a PCR-based test for hereditary hemochromatosis (HH).

The amino acid mutation C282Y, associated with HH in >80% of homozygous individuals, is caused by a single nucleotide polymorphism (A845G) in the HFE gene. This SNP creates an additional site for restriction endonucleaseRsa I.A 390 bp fragment of the HFE gene, spanning amino acid C282, was amplified using KAPA Blood PCR Mix B in 50 µl reactions containing 10% v/v whole EDTA blood. Amplified DNA was recovered from cellular debris by centrifugation and digested directly with Rsa I. RE digestion products were electrophoresed in a 2.5% TBE-agarose gel and detected by ethidium bromide staining.

PCR products generated from the blood of homozygous wild-type (WT), homozygous mutant (M) and heterozygous (Het) individuals are shown on the left. RE digestion products (right) of the WT allele yields 2 fragments (250 bp + 140 bp), whereas the mutant allele yields 3 fragments (250 bp + 111 bp + 29 bp), of which the smallest is not detectable by ethidium bromide staining. Heterozygous individuals are identified by a digestion product consisting of the 250 bp, 140 bp and 111 bp fragments. Generated in collaboration with Unistel Medical Laboratories.

Direct amplification from the blood of non-human species

KAPA Blood PCR Kits may also be used for the direct amplification of DNA from the blood of non-human species, such as other mammals and birds, thereby offering cost and time savings in PCR-based veterinary testing. Blood from other species may be collected in the same manner as human blood. The optimal amount of blood used in a KAPA Blood PCR must be determined empirically for each species. With mouse blood, best results have been obtained with a lower concentration of blood in the PCR (0.1 - 5% v/v) than recommended for humans. For bird species with nucleated erythrocytes, blood should be diluted to more closely approximate the DNA concentration in the same volume of human blood.

High-throughput genotyping of mice used in investigations into the relationship between metallothionein (MT) expression and mitochondrial function. MT knockout mice carry a short insertion in the MT gene on both alleles, which results in the absence of functional metallothionein. To distinguish mice carrying the knockout mutation on one or both alleles from homozygous wild-type mice, a fragment of the MT gene is amplified directly from mouse blood collected on “Guthrie cards”, using KAPA Blood PCR Kit A. A standard 3-step cycling protocol (30 cycles) with an initial denaturation time 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.

Frequently Asked Questions

1. What enzyme do KAPA Blood PCR Kits contain?
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 a process of high-throughput molecular 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.

KAPA Blood DNA Polymerase was evolved from a Type A polymerase and has 5’-3’ polymerase and 5’-3’ exonuclease activity, but no 3’-5’ exonuclease (proofreading) activity. The fidelity of KAPA Blood DNA Polymerase is similar to that of wild-type Taq. DNA fragments generated with KAPA Blood DNA Polymerase are A-tailed.

2. 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.

DNA fragments generated with either KAPA Blood PCR Kits may be digested with restriction endonucleases, sequenced or analysed using
dHPLC systems.

3. What are the applications best suited for PCR directly from whole blood?
KAPA Blood PCR Kits have been validated primarily for genetic testing and offer a viable alternative to amplification of DNA isolated from blood (using crude extraction methods of DNA purification kits), for the following applications:

• Single amplicon or multiplex end-point PCR, employing unlabelled or fluorescently labelled primers.
• SNP analysis, based on RE digestion of PCR products.
• Paternity testing using the PowerPlex16® System (Promega Corporation).

KAPA Blood PCR Kits may be used in protocols based on amplicons with a GC content <65% up to 3.5 kb in length and amplicons with a GC content <65% up to 1 kb in length.

4. What are the major benefits of using KAPA Blood PCR Kits in genetic testing?
KAPA Blood PCR Kits eliminate the need to extract or purify DNA prior to genetic testing. This reduces:

• Cost.
• Turnaround time.
• The risk of sample cross-contamination.

5. What benefits do KAPA Blood PCR Kits offer in genetic testing if DNA has to be isolated for archiving purposes?
Even if sample DNA has to be isolated for archiving purposes, KAPA Blood PCR Kits still offer the benefits of reducing the risk of sample cross-contamination during test phase, as well as the turnaround time for test results.
 
DNA isolation for the purposes of archiving may be eliminated by collecting/storing samples on Whatman FTA® Cards or 903® Specimen Collection Paper (“Guthrie cards”).

6. Can KAPA Blood PCR Kits be used for pathogen detection directly in blood?
DNA fragments derived from pathogens have been successfully amplified 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.

7. Which type of blood samples may be used as templates in Whole Blood PCRs using KAPA Blood PCR Kits?
KAPA Blood PCR Kits have been validated for the amplification from DNA directly from the following samples:

• 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 PCRs.

8. Which type of blood samples are unsuitable as templates in Whole Blood PCRs using KAPA Blood PCR Kits?
KAPA Blood PCR Kits are not suitable for the direct amplification of DNA from 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.

9. 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 Q.7), has been achieved.

10. 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 PCRs depends on the reaction volume, amplicon type and application.
 
The optimal concentration of blood in KAPA Blood PCRs 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.

11. 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 MixesA and B have been optimally formulated 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.

12. 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 successfully amplified 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.

13. 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.

14. 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 min at maximum speed (14,000 - 17,000 x g) in a benchtop microcentrifuge 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 facilitaterecovery 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 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.

15. Are KAPA Blood PCR Kits dHPLC compatible?
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.

16. Does Whole Blood PCR with KAPA Blood PCR Kits require any pre-treatment 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 PCRs.

17. 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 theoriginal protocol with isolated DNA as template. However, please ensure that:

• The initial denaturation time is 5 - 10 min (94 - 95°C).
• The denaturation time in each cycle is at least 30 sec (94 - 95°C).
• The annealing time in each cycle is at least 30 sec (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.

18. Which reaction parameters are most likely to affect the outcome of a KAPA Blood PCR?
Poor results (e.g. failed amplification, low yields and/or non-specific amplification) may be obtained when:

• 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 have undergone too many freeze-thaw cycles. 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.