Accessing Genetic Accessing Genetic Variation: Genotyping Variation: Genotyping

Single Nucleotide Single Nucleotide PolymorphismsPolymorphisms

Syvänen, Nature Genetics Syvänen, Nature Genetics 2001:930-9422001:930-942

February 24, 2004February 24, 2004

What is Genotyping?What is Genotyping?

Currently, the focus has changed from Currently, the focus has changed from SNP discovery to genotyping individualsSNP discovery to genotyping individuals

Using genotyping we can correlate Using genotyping we can correlate linkage linkage disequilibrium (LD)- “Genes not in random disequilibrium (LD)- “Genes not in random association” Hartl and Clark -1997 (pg 96), in association” Hartl and Clark -1997 (pg 96), in order to find a correlation of multifactorial traits order to find a correlation of multifactorial traits to candidate genes.to candidate genes.

Genotyping follows two formats - the Genotyping follows two formats - the assay assay used as well as the used as well as the detectiondetection method method

Recent techniques used Recent techniques used to map locations using to map locations using

SNP genotypesSNP genotypes

This table demonstrates the number of SNPs and This table demonstrates the number of SNPs and samples necessary to do population genetics or samples necessary to do population genetics or associate linkage disequilibriumassociate linkage disequilibrium

Flow chart for Flow chart for GenotypingGenotyping

Hybridization MethodsHybridization Methods

Hybridization of probe is extremely Hybridization of probe is extremely stringent at optimal conditions (false stringent at optimal conditions (false positives)positives)

Semi-homogenous (Chemiluminescence Semi-homogenous (Chemiluminescence or fluorescence)or fluorescence)

Solid-phase microarrays (Fluorescence)Solid-phase microarrays (Fluorescence) Solution-phase homogeneous Solution-phase homogeneous

(Fluorescence polarization or FRET)(Fluorescence polarization or FRET)

How does hybridization How does hybridization work with Real-Timework with Real-Time

TaqMan probeTaqMan probe

Molecular Beacon probeMolecular Beacon probe

Allele Fluorescence profileAllele Fluorescence profile

Real Time DetectionReal Time Detection

This figure gives a This figure gives a better overview of better overview of how both TaqMan how both TaqMan and Molecular and Molecular Beacons bind at a Beacons bind at a site and release site and release fluorescence, if the fluorescence, if the SNP corresponds SNP corresponds to the probeto the probe

Great for Great for multiplexingmultiplexing

TaqManTaqMan Molecular BeaconMolecular Beacon

Kirk Kirk et al.et al. 2002 (Fig 2) 2002 (Fig 2)

Primer ExtensionPrimer Extension

Follows every Follows every assay format assay format except solid-phase except solid-phase microarraysmicroarrays

Microarray primer Microarray primer extensionextension

Can detect Can detect heterozygosityheterozygosity

Potential rate of Potential rate of false positivesfalse positives

Primer ExtensionPrimer Extension PyrosequencingPyrosequencing - uses - uses

pyrophoshates, apyrase, pyrophoshates, apyrase, and luciferase to and luciferase to measure the exact base measure the exact base pair sequence pair sequence incorporated after the incorporated after the sequencing primersequencing primer

Can get up to 50 base Can get up to 50 base pairspairs

Can not multiplex, plus Can not multiplex, plus very costlyvery costly

www.pyrosequencing.comwww.pyrosequencing.com

Whether ligation occurs, Whether ligation occurs, depends on SNPdepends on SNP

If probes matched perfectly, If probes matched perfectly, then they will be joined with a then they will be joined with a ligase, in which the SNP can be ligase, in which the SNP can be categorizedcategorized

Potential multiplexing Potential multiplexing capabilitiescapabilities

Solid-phase microtiter plate Solid-phase microtiter plate (indirect colorimetric or FRET)(indirect colorimetric or FRET)

Allele-specific ligation Allele-specific ligation probesprobes

Allele-specific PCRAllele-specific PCR

Uses primary allele Uses primary allele primer and primer and secondary probe to secondary probe to create fluorescence create fluorescence for SNP detectionfor SNP detection

Primers very Primers very expensiveexpensive

Based on the idea Based on the idea of FRET of FRET (Fluorescence (Fluorescence Resonance Energy Resonance Energy Transfer)Transfer)

Endonuclease CleavageEndonuclease Cleavage

Invader probeInvader probeRFLPRFLP

Invader probe helps cleavage by Flap endonuclease, which emits Invader probe helps cleavage by Flap endonuclease, which emits fluorescence detected by Mass Specfluorescence detected by Mass Spec

Uses solution-phase homogeneous (Fluorescence polarization or Uses solution-phase homogeneous (Fluorescence polarization or FRET)FRET)

ConclusionConclusion Each technique has its own unique ability to Each technique has its own unique ability to

detect SNPs depending on the assay or detect SNPs depending on the assay or detection methoddetection method

One must weigh factors such as:One must weigh factors such as: CostCost ThroughputThroughput Simplicity of assay designSimplicity of assay design MultiplexingMultiplexing Sensitivity/accuracySensitivity/accuracy

After all these factors are analyzed then you After all these factors are analyzed then you can pick the correct method for your SNP can pick the correct method for your SNP detectiondetection