Techniques of SNP Genotyping can be summarized as follows: There are several techniques for genotyping SNPs including hybridization methods, enzyme-based methods, and other methods based on physical properties of DNA. Popular hybridization methods include DASH, molecular beacons, and gene chip arrays. Common enzyme-based techniques are RFLP, Invader assay, and oligonucleotide ligation assay. Other physical property-based methods include SSCP, TGGE, and pyrosequencing. Each method has its own pros and cons related to factors like speed, cost, and accuracy. Choosing the appropriate SNP genotyping technique depends on the number of SNPs needed to be analyzed and sample size.

1. Techniques of SNP Genotyping

2. SNPs
Single
Nucleotide
Polymorphisms
Single Nucleotide variation at a specific locationSingle Nucleotide variation at a specific location
in the genome
SNP: Commonly biallelic
C T Commonly found

3. 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

4. Popularity of different Polymorphism studies

5. SS
NN
PP
--
DD
AA
TTTT
AA
BB
AA
SS
EE

6. 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 the
world today.
With 3 billion nucleotides = each
nucleotide in the world today is mutated
340 times.

7. What can SNPs tell you?
Linkage Disequilibrium
Recombination
Association Studies
Demographic eventsDemographic events

8. Linkage Disequilibrium
The non-random association between
alleles in a population.
No LD
2 SNPs 4 Haplotypes
High LD
2 SNPs 2 Haplotyps

9. Association studies
An association between a genetic variant and a
phenotype.
Two groups: Disease Group Control (random pop)
Look for a variant which is in high freq in your disease
group in respect to the control.
Cystic Fibrosis was the first successful association
study, based on RFLPs. (Kerem et al Science 1989)
SNP at Promoter (-169C- susceptible/T non
susceptible) region of FCRL3 for Rheumatoid arthritis
in Japanese population (Kochi et al., Nat Genet. 2005
Jun; 37(6):652.)

10. SNP typing Techniques
Hybridization
Methods
Enzyme based
Methods
Other methods
based on physical
Properties of DNA
1. DASH
2. Molecular beacons
1. RFLP
2. Invader Assay
1. 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

11. DASH
PCR is performed using one of the primers contains a 5’
biotin.
Resulting products immobilized by a streptavidin-coated
microtiter plate.
The non-biotynylated strand is removed by NaOH
solution.
Hybridized with an oligonucleotide probe, (hybridization
buffer containing a fluorescent double-strand-specificbuffer containing a fluorescent double-strand-specific
intercalating dye).
The sample is heated slowly from room temperature to
above denaturing temperature while continually
monitoring fluorescence.
By plotting the negative derivative (slope of the
fluorescence Vs temp.), denaturation points are clearly
seen as peaks. Peak temperature values can be used
for final allele determination.

12. DASH

13. Molecular Beacons

14. Genomic DNA
PCR amplified SNP
region
RE Digestion SNP-RFLPRE Digestion
Gel electrophoresis
SNP genotyping
SNP-RFLP
Genotyping

15. Invader assay
3’ 5’3’ 5’

16. Oligonucleotide ligation Assay

17. Taqman assay

18. Taqman assay

19. Primer extension (CPE- Mass analysis)
Mass analysis
MALDI-TOF MS
Commercial SNP assays:
Pin pint assay
Mass EXTEND
SPC-SBE
GOOD assay

20. CPE- Fluorescence analysis
Commercial SNP assays:
SNaPshot approach (Applied Biosystems)
SNP stream assay (Orchid Biosciences)
Fluorescence analysis

21. Primer extension (SPE- Mass analysis)
Tag-It approach (Tm Bioscience corp, Canada)

22. SSCP
Genomic DNA
PCR amplification
Denatured by heat/Denatured by heat/
formaldehyde
Run on non-
denaturing
electrophoresis gel

23. Pros & cons
PROS
Rapid
Technically simple
Inexpensive
Uses commonly available equipment
Detects 60-95% of mutations in short DNA strandsDetects 60-95% of mutations in short DNA strands
CONS
Need to sequence DNA to identify specific mutation
ssDNA conformation is highly dependent on temperature and
it is not generally apparent what the ideal temperature is.
Sensitivity drops when sequences longer than 400 bp are
used (Costabile et al. 2006).

24. 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

26. Invader assay- Third wave technologies

27. Genechip array- Affymetrix
Allele-specific probes
consisting of 25-mer
oligonucleotides are
synthesized in an orderedsynthesized in an ordered
fashion to form a probe array

28. Pyrosequencing
Mix genomic DNA with PCR reagents & thermocyle
Purify biotinylated PCR products using Streptavidin-Sepharose
Anneal extension primer to single stranded biotinylated PCR
product
Pyrosequencing reaction
This methodology is good for
small numbers of SNPssmall numbers of SNPs
(less than 15)
and on small
sample sizes (1-1000)

32. Thank You … !Thank You … !