Techniques of SNP Genotyping

SNPs

Single

Nucleotide

Polymorphisms

�Single Nucleotide variation at a specific location �Single Nucleotide variation at a specific location

in the genome

�SNP: Commonly biallelic

�C T Commonly found

Regulatory sites

Coding Regions

Exons

Introns Non coding region

3’ end5’ end

SNP: Alterations in

protein structure

Non coding region

Regulatory sites

(Promoter)

Introns Non coding region

SNP:

�Transcription rate

changes

�Encoded protein

production

changes

�High frequency

�Evolutionarily stable

Unique in particular geographical or ethnic group

�important markers for comparative and

Evolutionary genomics studies

Popularity of different Polymorphism studies

SS

NN

PP

--

DD

AA

TTTT

AA

BB

AA

SS

EE

How many SNPs are there in Humans today?

� Human Mutation rate is ~2.5 x 10-8

mutations/site/gen

� ~150 mutations/diploidgenome/generation

� 6.8 billion people in the world=1,020,000,000,000 mutations in the=1,020,000,000,000 mutations in theworld today.

� With 3 billion nucleotides = eachnucleotide in the world today is mutated340 times.

What can SNPs tell you?

� Linkage Disequilibrium

� Recombination

� Association Studies

� Demographic events� Demographic events

Linkage Disequilibrium

� The non-random association between

alleles in a population.

No LD

2 SNPs 4 Haplotypes

High LD

2 SNPs 2 Haplotyps

Association studies

� An association between a genetic variant and aphenotype.

� Two groups: Disease Group Control (random pop)

� Look for a variant which is in high freq in your diseasegroup in respect to the control.

� Cystic Fibrosis was the first successful associationstudy, based on RFLPs. (Kerem et al Science 1989)

� SNP at Promoter (-169C- susceptible/T nonsusceptible) region of FCRL3 for Rheumatoid arthritisin Japanese population (Kochi et al., Nat Genet. 2005Jun; 37(6):652.)

SNP typing Techniques

Hybridization

Methods

Enzyme based

Methods

Other methods based on physical Properties of DNA

1. DASH

2. Molecular beacons1. RFLP

2. Invader Assay1. SSCP

2. TGGE

Some commercial

techniques

3. Primer Extension

4. Oligonucleotide ligation assay

5. Taqman assay

2. TGGE

1. Gene chip array- Affymetrix

2. Bead array- Illumina

3. Pyrosequencing – Illumina

DASH

� PCR is performed using one of the primers contains a 5’biotin.

� Resulting products immobilized by a streptavidin-coatedmicrotiter plate.

� The non-biotynylated strand is removed by NaOHsolution.

� Hybridized with an oligonucleotide probe, (hybridizationbuffer containing a fluorescent double-strand-specificbuffer containing a fluorescent double-strand-specificintercalating dye).

� The sample is heated slowly from room temperature toabove denaturing temperature while continuallymonitoring fluorescence.

� By plotting the negative derivative (slope of thefluorescence Vs temp.), denaturation points are clearlyseen as peaks. Peak temperature values can be usedfor final allele determination.

DASH

Molecular Beacons

Genomic DNA

PCR amplified SNP

region

RE Digestion SNP-RFLP RE Digestion

Gel electrophoresis

SNP genotyping

SNP-RFLP

Genotyping

Invader assay

3’ 5’3’ 5’

Oligonucleotide ligation Assay

Taqman assay

Taqman assay

Primer extension (CPE- Mass analysis)

Mass analysis

MALDI-TOF MS

Commercial SNP assays:

Pin pint assay

Mass EXTEND

SPC-SBE

GOOD assay

CPE- Fluorescence analysis

Commercial SNP assays:

SNaPshot approach (Applied Biosystems)

SNP stream assay (Orchid Biosciences)

Fluorescence analysis

Primer extension (SPE- Mass analysis)

Tag-It approach (Tm Bioscience corp, Canada)

SSCP

Genomic DNA

PCR amplification

Denatured by heat/ Denatured by heat/

formaldehyde

Run on non-

denaturing

electrophoresis gel

Pros & cons

PROS

� Rapid

� Technically simple

� Inexpensive

� Uses commonly available equipment

� Detects 60-95% of mutations in short DNA strands� Detects 60-95% of mutations in short DNA strands

CONS

� Need to sequence DNA to identify specific mutation

� ssDNA conformation is highly dependent on temperature andit is not generally apparent what the ideal temperature is.

� Sensitivity drops when sequences longer than 400 bp areused (Costabile et al. 2006).

C

G

G

C

C

G

G

GG

C

C

A

A

T

Normal DNA

Target DNA

Denaturing followed by annealing

Homoduplexes Homoduplexes Heteroduplexes

C

G C

A T

T

Homoduplexes Homoduplexes Heteroduplexes

TGE TGE

TGE

Invader assay- Third wave technologies

Genechip array- Affymetrix

Allele-specific probes

consisting of 25-mer

oligonucleotides are

synthesized in an ordered synthesized in an ordered

fashion to form a probe array

Pyrosequencing

� Mix genomic DNA with PCR reagents & thermocyle

� Purify biotinylated PCR products using Streptavidin-Sepharose

� Anneal extension primer to single stranded biotinylated PCRproduct

� Pyrosequencing reaction

� This methodology is good for

small numbers of SNPssmall numbers of SNPs

(less than 15)

and on small

sample sizes (1-1000)

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