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Single Nucleotide Polymorphism Genotyping Using

Kompetitive Allele Specific PCR (KASP)

Manglam Arya

Msc.(Ag.) Biotechnology

CPBMB, COH.

Single Nucleotide Polymorphism

• Single nucleotide polymorphism (SNP)

refers to a single base change in a DNA

sequence

• SNP: Commonly biallelic

• Two types(Based on presence in

genome)

Synonymus

Non-synonymus

• SNPs have largely replaced simple

sequence repeats (SSRs)

Advantage of using SNPs

Low assay cost

High genomic abundance

Locus specificity

co-dominant inheritance

Simple documentation

Potential for high-throughput Analysis

Relatively low genotyping error rates

• BeadXpressTM,GoldenGateTM

and Infinium from Illumina

• GeneChipTM and GenFlexTM Tag

array from Affimetrix

• SNaPshotTM and TaqManTM

from the Applied Biosystems

• SNPWaveTM from KeyGene

• iPLEX GoldTM Assay and Mass-

RRAYTM from Sequonome

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• SNPstreamTM from BeckmanCoulter

• Pyrosequencing from RoyalInstitute of Technology (Sweden)

• Molecular inversion probes fromParAllele Biosciences

• Competitive allele-specific PCR(currently called KompetitiveAllele Specific PCR, or KASPTM)from Kbioscience or LGCGenomics

SNP genotyping platforms

Variables to be considered

Throughput

Data turnaround

Time

Ease of use

Performance (sensitivity, reliability, reproducibility, and

accuracy),

Flexibility (genotyping few samples with many snps or

many samples with few snps),

Number of markers generated per run (uniplex versus

multiplex assay capability)

Assay development requirements and genotyping cost per

sample or data point.5

KASP

KBioscience Competitive Allele-Specific PCR

Homogenous, Fluorescence-based genotyping technology, based on

Allele-specific oligo extension (primer)

Fluorescence resonance energy transfer

Determine both SNP and insertion/deletion

genotypes

Analysis can be carried out in 96-, 384- and 1536-

well plate formats

1)Purified DNA sample (5-10ng)

2)Two allele-specific forward primers(Each primer contains a unique

unlabelled tail sequence at the 5' end)

3) A common reverse primer

4)Two 5’ Fluro ‐labelled oligos, one labelled with FAM(Fluorescein

Amidite) and another with HEX

(FERT- Fluorescent Resonance Energy Transfer)

5) Two oligos, with quenchers bound at the 3‘ ends.

Working of KASP

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Bi-allelic discrimination is achieved

through the competitive binding of the two

allele-specific forward primers

If the genotype is homozygous, only one

of the two possible fluorescent signals will

be generated

If the genotype is heterozygous, a mixed

fluorescent signal will be generated

KlusterCaller Software

When dual emission genotyping data

from a fluorescent reader is imported

into the KlusterCaller software, a

traditional cluster graph for each assay is

automatically generated

For each KlusterCaller project, a project

tree will be created on the left hand side

of the window

Within a DNA master plate map,

individual wells can be identified as

DNA samples, no template controls

(NTC), or empty.

Genotyping data that is imported into a

KlusterCaller project will be displayed as a

Cartesian cluster plot

FAM values are plotted on the X axis and

HEX values are plotted on the Y axis,

Normalisation of results using ROX

(passive reference dye)

Blue data points are homozygous for the

allele reported by FAM, green data points

are heterozygous and red data points are

homozygous for the allele reported by

HEX

The black data points represent the notemplate controls (NTC) and pink datapoints are unconfirmed

KlusterCaller Software

GoldenGate assay KASP

Golden Gate v/s KASP

Genotyping cost comparisons between the KASP,

BeadXpress and GoldenGate platforms

KASP Applications

• Genotyping a wide range of species for various

purposes.

• KASP for Quality analysis, QTL mapping,

MARS, and allele mining

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Quality Control Analysis

• QC analysis should be done for two reasons bygenotyping the parents and F1s with the samesubset of SNPs, in order to

confirm if F1s contains true-to-type alleles fromtheir parents

check the genetic purity of the inbred parents.

• F1s with true-to-type parental alleles for at least 90% of the SNPs that were polymorphic between theparents should be advanced, while those with lessthan 10 % nonparental alleles should be discarded.

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QTL Mapping

QTL mapping identifies a subset of markers that are significantly associated with one or more QTL influencing the expression of the trait of interest.

1) Select or develop a bi-parental mapping population.

2) Phenotype the population for a trait under greenhouse or field conditions.

3) Choose a molecular marking system – genotype parents of the mapping population and F1s with large numbers of markers, then select 200-400 markers exhibiting polymorphism between the parents.

4) Choose a genotyping approach, then generate molecular data for polymorphic markers

5) Identify the molecular markers associated with major QTL using statistical programs.

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• Allele mining is a promising approach to dissecting

naturally occurring allelic variation at candidate genes

controlling key agronomic traits.

• KASP platform at CIMMYT has been used for the

systematic mining of large germplasm collections for

specific functional polymorphisms.

• SNPs or small indels that are either diagnostic or tightly

linked to such polymorphisms can be used in the KASP

platform to query a large germplasm collection to identify

accessions with favorable alleles at the target locus/loci

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Large-scale allele mining

Reference

• www.cerealsdb.uk.net

• www.ksre.ksu.edu

• www.lgcgroup.com/_LGCGroup_media_PDFs_Products_Genotyping_KA

SP-genotyping-chemistry-User-guide.pdf_ext

• Semagn, K ., Raman Babu, Hearne, S. and Olsen, M. 2014. Single

nucleotide polymorphism genotyping usingKompetitive Allele

Specific PCR (KASP): overview of the technology and its

application in crop improvement. Mol Breeding 33:1–14

• http:/en.wikipedia.org/wiki/SNP_GENOTYPE

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