snp genotyping technologies
Post on 03-Jun-2015
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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)
Thank You … !Thank You … !
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