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 SNP genotyping platforms 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 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. KASP KBioscience Competitive Allele-Specific PCR Homogenous, Fluorescence-based genotyping technology, based on Allele-specific oligo extension (primer) Fluorescence resonance energy transfer KASP Applications Genotyping a wide range of species for various purposes. KASP for Quality analysis, QTL mapping, MARS, and allele mining Quality Control Analysis QC analysis should be done for two reasons by genotyping the parents and F1s with the same subset of SNPs, in order to confirm if F1s contains true-to-type alleles from their 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 the parents should be advanced, while those with less than 10 % nonparental alleles should be discarded. 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. Large-scale allele mining 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

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Single Nucleotide Polymorphism Genotyping Using
Kompetitive Allele Specific PCR (KASP)
Manglam Arya
Msc.(Ag.) Biotechnology
CPBMB, COH.

2. 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)

3. 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

4. • 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
4
• SNPstreamTM from Beckman
Coulter
• Pyrosequencing from Royal
Institute of Technology (Sweden)
• Molecular inversion probes from
ParAllele Biosciences
• Competitive allele-specific PCR
(currently called Kompetitive
Allele Specific PCR, or KASPTM)
from Kbioscience or LGC
Genomics
SNP genotyping platforms

5. 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

6. 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

7. 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

8. 8

11. 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

12. 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.

13. 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 no
template controls (NTC) and pink data
points are unconfirmed
KlusterCaller Software

14. GoldenGate assay KASP
Golden Gate v/s KASP

15. Genotyping cost comparisons between the KASP,
BeadXpress and GoldenGate platforms

16. KASP Applications
• Genotyping a wide range of species for various
purposes.
• KASP for Quality analysis, QTL mapping,
MARS, and allele mining
16

17. Quality Control Analysis
• QC analysis should be done for two reasons by
genotyping the parents and F1s with the same
subset of SNPs, in order to
confirm if F1s contains true-to-type alleles from
their 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 the
parents should be advanced, while those with less
than 10 % nonparental alleles should be discarded.
17

18. 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.
18

19. • 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
19
Large-scale allele mining

20. 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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