Page 1
KASP
genotyping
technology
For Research Use Only.
Not for use in diagnostic procedures.
Page 2
An introduction
How KASP works
to KASP
KASP™ genotyping technology from
LGC, Biosearch Technologies™ utilises a
unique form of competitive allele-specic
PCR (polymerase chain reaction) that
enables highly accurate bi-allelic scoring of
SNPs (single nucleotide polymorphisms)
and indels (insertions/deletions) at
specic loci across a wide range of
genomic DNA samples, including those
of complex genomes. KASP technology
delivers extremely high levels of assay
robustness and accuracy with signicant
cost savings.
Superb accuracy
and performance
• Accuracy >99.8% based on independent
assessment
• Industry leading SNP and InDel assay
conversion rate (>90%).
Breakthrough simplicity
and exibility
• KASP uses a universal reporting system,
which simplies assay design and eliminates
time-intensive probe design steps
• Requires only 10 ng DNA per sample per SNP
(based on human genome size)
• Low reaction volumes keep reagent costs to a
minimum
• Cost benets enable you to perform more
assays overall, improving the quality of your
data.
KASP genotyping chemistry is available as a
service in Europe and North America or as
in silico wet lab validated and non-validated kits.
1. Assay components:
A) KASP Assay Mix: consists
of 2 allele specic primers and
1 reverse primer.
B) KASP-TF Master Mix:
contains universal uorescent
probes, Taq polymerase and
dNTP‘s in an optimised buffer
solution.
C) Sample DNA: DNA
contains the SNP of interest.
A) KASP Assay Mix
Allele-specic forward primers:
allele-1
5′
allele-2
5′
Reverse primer:
5′
3′
B) KASP-TF
Master Mix
C
3′
A
3′
5′3′
C) DNA template (sample)
G/T
2. Denatured template and annealing
components – PCR round 1:
(allele-2 primer does not elongate)
A
(allele-1 primer binds and elongates)
5′
3′
In the rst round of PCR, one of the allele-specic primers matches the target
SNP and with the common reverse primer, amplies the target region.
5′ 3′
(reverse primer elongates 5′-3′)
C
G
3. Complement of allele-specic tail sequence
generated – PCR round 2:
F Q
3′
5′
5′
3′
Legend
Allele-1 tail FAM-labelled
oligo sequence
Allele-2 tail HEX-labelled
oligo sequence
Common reverse primer
FAM dye
F
HEX dye
H
Target SNP
Quencher
Q
H Q
3′
5′
5′
3′
′
3
′
5
Tremendous exibility
• Flexible primer design which increases the
rate of successful assay development
• Supports low-, medium- and high-throughput
studies and individual repeat assays
• Compatible with a wide range of liquid handling
systems and thermal cyclers;
signal can be read on most FRET
(uorescent resonant energy transfer)
capable plate readers and qPCR
machines.
2
KASP by Design
(KBD)
Designed in silico
No validation
KASP on Demand
(KOD)
Designed in silico
Optimised and validated
Assembled as a homogeneous assay in 96-,
384- or 1536-well PCR plates, bi-allelic
discrimination is achieved through the competitive
binding of two allele-specic forward primers,
each with a unique tail sequence that
corresponds with one of two universal probes;
one labelled with FAM™ dye and the other with
HEX™ dye.
C
G
4. Signal generation – PCR round 3:
FAM-labelled oligo binds to new complementary
F
In further rounds of PCR, levels of allele- specic tail increase. The
uor labelled part of the FRET cassette is complementary to new tail
sequences and binds, releasing the uor from the quencher to generate
a uorescent signal.
tail sequence and is no longer quenched.
C
G
(Reverse primer binds,
elongates and makes a
complementary copy of
the allele-1 tail.)
Q
5′
Fluor for incorporated
G allele no longer
quenched.
3′
Fluor for non-incorporated
T allele remains
quenched.
H Q
3′
5′
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KASP
genotyping
assay
components
Designing a KASP genotyping assay is fast and
easy. KASP uses a universal reporting system
that eliminates the need to design assay-specic
uorescent probes. Simply create your Assay mix
consisting of your assay-specic primers, add your
DNA sample and KASP-TF Master Mix. You can
run KASP in 96-, 384- or 1536-well PCR plates.
To run, seal the plate and run your PCR cycling
protocol. At the end of the thermal cycle, conduct
an end-point PCR read using either a qPCR
instrument or FRET-capable plate reader. No
separation by agarose gel is required.
2. KASP-TF Master
1. KASP Assay Mix
The KASP Assay Mix contains three assay-specic
non-labelled primers: two allele-specic forward
primers plus one common reverse primer. The
sequences for these primers are generated via our
proprietary Kraken™ software. KASP Assay Mix can
be purchased in two formats:
1. KASP by Design (KBD)
– designed in silico
2. KASP on Demand (KOD)
– validated and optimised in our laboratories.
KOD assays are guaranteed to generate
accurate genotyping data when used in
conjuction with DNA of suitable quality and
KASP-TF Master Mix.
KASP Assay Mix is supplied in a single 2D
barcoded tube containing sufcient Assay mix to
run a minimum of 2500 x 10 µL reactions - the
recommended volume for 96-well PCR plates. If run
in 384-well PCR plates, the total reaction volume can
be reduced to 5 µL, doubling the number of reactions
and cutting the cost per data point in half.
For breakthrough savings, customers can utilise our
SNPline workow for high-throughput PCR, that we
developed for use in our own service laboratories
and that enables 1 µL reactions in 1536-well PCR
plates. SNPline workow customers can achieve
a 10-fold reduction in the cost per data point, while
increasing throughput up to 500,000 data points per
day.
KASP
genotyping
workow
Assembly of KASP genotyping reactions in 96-, 384- or 1536-well PCR plates
(total reaction volumes from 10 μL down to 1 μL)
DNA samples
• DNA extracted from
plant, animal, human
or microbial species
• 10 ng of DNA required
per reaction (based on
human genome size)
Heat/laser/pressure sealing of plates
Cluster analysis - assign genotypes to samples
KASP Assay Mix
• Three target specic
primers per assay
(no labelling required)
- Two allele-specic
forward primers
- One reverse primer
• Each SNP or InDel
assay provided in a
single 2D barcoded tube
PCR thermal cycle
Fluorescent reading of plates
Data export
KASP-TF Master Mix
• FRET reporting system
(universal FAM and
HEX labelled cassettes)
• DNA polymerase
• ROX passive
reference dye
• dNTPs
• MgCl
• Optimised buffer
2
Mix
KASP-TF Master Mix is formulated without Triton™
X-100 (Triton-Free), and contains the universal
FRET cassettes, ROX™ passive reference dye,
KASPTaq™ DNA polymerase, free nucleotides and
MgCl2 in an optimised buffer solution. KASP-TF
Master Mix is universal to every KASP genotyping
assay and is offered in a range of standard
packaging sizes to address varying throughput
needs. Custom packaging solutions are also
available if needed.
4
KASP
Assay Mix
KASP-TF
Master Mix
KASP
genotyping
reaction
Sample
DNA
Following completion of the KASP
PCR, reaction plates are read and the
data analysed using any cluster analysis
viewing software. Detected signals are
plotted as a graph, with samples of the
same genotype clustering together. In
our genotyping service laboratories,
cluster analysis is performed using
our proprietary Kraken software. An
example of the data generated is
shown on the right.
Red
Green
Blue
Sample is
homozygous for
the HEX allele
Sample is
heterozygous:
one FAM allele
and one HEX allele
Sample is
homozygous for
the FAM allele
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Page 4
Request
a Free KASP Trial Ki
KASP products
KASP products
Ordering
Request a free
and services
and services
Product options Service
KBD -
KASP by Design
Relative cost and value provided + ++ +++
Lead times 1-3 weeks
Customer submits reference sequences Yes Yes Yes
Target-specic KASP primer sequences are
designed for each assay using proprietary
Kraken software
Target specic primer sequences and
common reverse primers prepared
Primers pooled into a single
2D barcoded tube to create the
KASP Assay Mix
Assay validation is performed in our service
lab with customer supplied DNA. Should
any assay fail, assay conditions and/or
primer sequences will be optimised until the
assay works
Biosearch Technologies provides a
money-back guarantee that each assay will
work
Biosearch Technologies service lab runs
customer samples and provides genotyping
data
1
Our proprietary Kraken software has an industry leading SNP-to- assay conversion rate that exceeds 90%. This means that, on average, 9 out of 10 KBD
assays will wor k based on standard conditions, without any optimisation.
2
Once validation has been completed for a particular SNP, it does not need to be repeated. If a customer orders a set of assays as KOD products, the next time
they order this same set, they would order the assays as KBD products since the validation work has already been completed. The same holds true for ser vice
orders; the set-up charge applies only to the rst order.
standard conditions)
Yes Yes Yes
Yes Yes Yes
Yes Yes Yes
No1
( >90% of assays
work based on
No Yes No
No No Yes
KOD -
KASP on Demand
Standard: 4-6 weeks
Fast: 1-3 weeks
Yes 2
(Validation testing
accounts for KBD/KOD
price variance)
Genotyping service using
KASP
Quoted based on scope
(# of SNPs/# of samples)
Typically 4- 6 weeks
Yes 2
(Validation testing captured
as set-up charge – this
charge is not applicable on
repeat orders)
information
KASP trial kit at:
biosearchtech.com/
kasp-free-trial-kit
KASP Assay Mix
Catalogue number Description and quantity
KBS-2300-001 KASP by Design Primer Mix, 2500 × 10 μL Reactions, Standard Service
KBS-2300-002 KASP by Design Primer Mix, 2500 × 10 μL Reactions, Standard Service, Manual Design
KBS-2400-001 KASP on Demand Primer Mix, 2500 × 10 μL Reactions, Standard Service
KBS-2400-002 KASP on Demand Primer Mix, 2500 × 10 μL Reactions, Rapid Service
KBS-2400-003 KASP on Demand Primer Mix, 2500 × 10 μL Reactions, Standard Service Manual Design (3 Primers)
KASP-TF Master Mix examples
Catalogue number Description and quantity
KBS-1050-101 KASP-TF V4.0 2X Master Mix 96/384, Standard ROX (2.5 mL)
KBS-1050-102 KASP-TF V4.0 2X Master Mix 96/384, Standard ROX (25 mL)
KBS-1050-103 KASP-TF V4.0 2X Master Mix 96/384, Standard ROX (250 mL) (10 × 25 mL)
KBS-1050-111 KASP-TF V4.0 2X Master Mix 1536, Standard ROX (2.5 mL)
KBS-1050-112 KASP-TF V4.0 2X Master Mix 1536, Standard ROX (25 mL)
KBS-1050-113 KASP-TF V4.0 2X Master Mix 1536, Standard ROX (250 mL) (10 × 25 mL)
KBS-1050-164 KASP-TF V4.0 Trial Kit (Standard ROX)
ROX is added to KASP-TF Master Mix as a passive reference dye. Different qPCR instruments have differing requirements for ROX; please
contact [email protected] to determine the correct version for your instrument. Formulations for use with 1536-well PCR plates are
compatible with SNPline™ instrumentation. Please enquire for ordering information.
Software and barcode reading
Catalogue number Description and quantity
KBS-0101-002 KlusterCaller, genotyping calling software
KBS-0101-001 Kraken full LIMS system for genotyping (site license)
KBS-0025-001 Cyclops 2D single tube reader
KBS-0025-002 ZTS-01 96-rack 2D reader
For Research Use Only. Not for use in diagnostic procedures.
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Integrated tools.
Accelerated science.
@LGCBiosearch
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