Banoth Madhu: SNP Genotyping Techniques and Epigenetic Markers. SNP and SNV genotyping are techniques that analyze genomic sequence variations. These single-base substitutions are typically detected using real-time PCR, microarrays, or next-generation sequencing (NGS) techniques. A single nucleotide variant (SNV) is a variation of a single nucleotide in a population’s genome.

1. SNP Genotyping Techniques and
Epigenetic Markers
Banoth Madhu
Ph.D Research Scholar
Genetics and Plant Breeding
TNAU, Coimbatore

2. SNP GENOTYPING 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
1. Gene chip array- Affymetrix
2. Bead array- Illumina
3. Pyrosequencing – Illumina

3. DASH

4. MOLECULAR BEACONS

5. GENOMIC DNA
PCR amplified SNP
region
RE Digestion SNP-RFLP
Gel electrophoresis
SNP genotyping
Genotyping

6. INVADER ASSAY
3’ 5’

7. OLIGONUCLEOTIDE LIGATION ASSAY

8. TAQMAN ASSAY

9. PRIMER EXTENSION (MASS ANALYSIS)
MALDI-TOF MS
Mass analysis
Commercial SNP assays:
Pin pint assay
Mass EXTEND
SPC-SBE
GOOD assay

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

11. C
G
G
C
G
G
G C
C
A
A
T
Denaturing followed by annealing
C G
Normal DNA
Target DNA
Homoduplexes
C
G C
Homoduplexes
A
Heteroduplexes
T
T
TGE TGE
TGGE

12. GENECHIP ARRAY-AFFYMETRIX
Allele-specific probes
consisting of 25-mer
oligonucleotides are
synthesized in an ordered
fashion to form a probe array

13. 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 SNPs
(less than 15)
and on small
sample sizes (1-1000)

14. EPIGENETIC MARKERS

15. EPIGENETIC MARKERS

16. EPIGENETIC MARKERS

17. EPIGENETIC MARKERS

18. EPIGENETIC MARKERS

19. EPIGENETIC MARKERS

20. EPIGENETIC MARKERS