highthroughput genotyping

Finnish Genome Center,Click to edit Master title style,*,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,22 September 2024,1,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Finnish Genome Center,*,High-throughput,genotyping,Finnish Genome Center,What is genotyping?,the analysis of DNA-sequence variation,genotype = the genetic constitution of an individual,2,Finnish Genome Center,Alleles,alternative form of a gene or DNA sequence at a specific chromosomal location (locus),at each locus an individual possesses two alleles, one inherited from the father one from the mother, genotype: a sum of these two alleles,3,Finnish Genome Center,Genetic differences:,may cause or predispose to diseases,determines e.g. individual drug response,used as markers to identify predisposing genes for diseases, high-throughput genotyping technologies needed,4,Finnish Genome Center,Microsatellite markers,Di-, tri-, tetranucleotide repeats,GAACGTACT,CACACACACACACA,TTTGAC,TTCGATGATA,GATAGATAGATAGATA,CGT,5,Finnish Genome Center,.Microsatellite markers,also called:,STR,=,S,ingle,T,andem,R,epeat,SSR,=,S,imple,S,equence,R,epeat,SSLP,=,S,imple,S,equence,L,ength,P,olymorphism,the number of repeats varies (, 30),highly polymorphic,distributed evenly throughout the genome,easy to detect by PCR,6,Finnish Genome Center,SNP markers,single nucleotide variation,GTGGACGTGCTT,G/C,TCGATTTACCTAG,7,Finnish Genome Center,The most simple and common type of polymorphism,Highly abundant; every 1000 bp along human genome,Most SNPs do not affect on cell function,some SNPs could predispose people to disease,influence the individuals response to a drug,Widely used as genetic markers,.SNP markers,8,Finnish Genome Center,SNP genotyping methods,over 100 different approaches,Ideal SNP genotyping platform:,high-throughput capacity,simple assay design,robust,affordable price,automated genotype calling,accurate and reliable results,9,Finnish Genome Center,.SNP genotyping methods,PCR,discrimination between alleles:,allele-specific hybridization,allele-specific primer extension,allele-specific oligonucleotide ligation,allele-specific enzymatic cleavage,detection of the allelic discrimination:,light emitted by the products,mass,change in the electrical property,10,Finnish Genome Center,The Finnish Genome Center,Independent department of,University of Helsinki,Since 1998,National core facility for the genetic research of multifactorial diseases,Provides collaboration and genotyping service to scientist and research groups in Finland, also abroad,11,Finnish Genome Center,The Finnish Genome Center; Goals,help designing genetic studies,perform high-throughput genotyping,perform data analysis,training of scientists,adopt and develop new strategies & technologies,12,Finnish Genome Center,Research strategies,Genome wide scans,with microsatellite markers,400 dinucleotidemarkers, 10 cM spacing,Fine mapping,Project specific (microsatellite) markers,SNP genotyping,Primer extension methods (SNuPe and MassArray; MALDI-TOF), restriction enzyme methods, project specific markers,13,Finnish Genome Center,Genome Scan,genotyping the whole genome with 400 microsatellite markers at 10 cM interval,look for chromosomal regions with excess allele sharing,0,30,40,50,60,70,80,90,100,110,120,130,10,20,microsatellite markers at 10 cM distance,14,Finnish Genome Center,Fine mapping,candidate regions identified by a genome scan,candidate genes,microsatellite or SNP markers,verification of linkage results,15,Finnish Genome Center,Setting up PCR-reactions,16,Finnish Genome Center,Electrophoresis run,17,Finnish Genome Center,Genotypes,18,Finnish Genome Center,What the data looks like,MarkerWell IDSampleIDAllele1Allele2Size1Size2,19,Finnish Genome Center,SNP genotyping at FGC,PCR-RFLP,(restriction fragment length polymorphism),SNuPe,(Single nucleotide primer extension),MassARRAY; MALDI-TOF,(,M,atrix,A,ssisted,L,aser,D,esorption/,I,onization,T,ime-,o,f-,f,light mass spectrometry),20,Finnish Genome Center,PCR-RFLP,Reactions designed to produce products of different sizes after enzymatic cleavage,C analyte,T,analyte,228,94,size in bp,Undigested PCR product,243,21,Finnish Genome Center,SNuPe,primer extension reactions designed to produce differentially labelled products,analysis by capillary electrophoresis (MegaBACE),GGACCTGGAGCCCCCACC,GGACCTGGAGCCCCCACC,C,GGACCTGGAGCCCCCACC,T,Extendable primer,C analyte,T,analyte,-,C (blue),T (red),labelled nucleotide,22,Finnish Genome Center,MassARRAY System (MALDI-TOF),Primer extension reactions designed to generate different sized products,Analysis by mass spectrometry,C/T,G/A,GGACCTGGAGCCCCCACC,GGACCTGGAGCCCCCACC,C,GGACCTGGAGCCCCCACC,T,C,Extendable primer,C analyte,T,analyte,Mass in Daltons,G/A,ddCTP,dTTP,dATP,dGTP,23,Finnish Genome Center,Mass spectrometry multiplexing,24,Finnish Genome Center,Primer extension mass spectrometry,Advantages:,accurate,automated assay design,fast automated data collection,multiplexing capacity,Disadvantages:,expensive instruments, consumables,extensive post-PCR processing,25,Finnish Genome Center,SNP genotyping workflow at FGC,26,