|
KAPA Blood PCR Kits
A second-generation DNA polymerase evolved specifically
for Whole Blood PCR.
KAPA Blood PCR Kits contain a second-generation DNA
polymerase evolved specifically for DNA amplification
directly from blood, in a choice of two optimized,
easy-to-use PCR mixes.
The result is industry-leading performance:
Product Description
KAPA Blood PCR Kits contain KAPA Blood DNA
Polymerase, a second-generation enzyme derived
through a process of molecular evolution and the
first DNA polymerase engineered specifically for the
amplification of DNA directly from whole blood. The
enzyme is available in two optimized, easy-to-use 2x
PCR mixes, which contain all components required for
Whole Blood PCR, except primers and template
(blood). Using KAPA Blood PCR Kits, 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
cost of genetic testing.
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:
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 endonuclease Rsa 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.
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.
KAPA Blood PCR Kits
.............................................................................................................................................................................................
|
