The document discusses single nucleotide polymorphisms (SNPs), which are variations in DNA that can distinguish individuals and are prevalent in the human genome. It outlines various methods for SNP detection, including both global and targeted discovery techniques, and details specific technologies such as denaturing gradient gel electrophoresis and Muts protein-binding assays. Additionally, the applications of SNP detection in fields like agriculture and pharmacogenetics are highlighted.

1. Advance single nucleotides
polymorphism (SNP) detection
methods
Presented By: Aneela Rafiq

2. Introduction
• Single nucleotide polymorphisms are single base
variations between genomes within a species.
• There are at least 10 million polymorphic sites in
the human genome.
• SNPs can distinguish individuals from one another.

3. Technology for Global SNP Discovery
• SNP in every 1,000 bp of DNA when two human
genomes are compared.
• Restriction Fragment Length Polymorphisms
(RFLPs) were the first SNPs .
• But very laborious strategy.

4. Cont…
• late 1990’s, several favorable factors came
together to make global SNP discovery a
reality.
1. human genome project
2. cost and efficiency of DNA sequencing
improved
3. bioinformatics tools
4. the SNP consortium (TSC)

5. Technology for Targeted
SNP Discovery
• Targeted SNP discovery
focuses on most and SNP
in regions of interest.
• Recently many SNP
scanning technologies are
developed.
• Sequencing is the perfect
method but not cost
effective.

6. Denaturing Gradient Gel Electrophoresis
• Denaturation is highly depend on DNA
sequence.
In many cases a single nucleotide
change the melting point.
And change melting point shows unique
position on gel

9. Complete Instrument of DGGE

10. Chemical Cleavage Of Mismatch
Find SNP by
specific
chemicals
Potassium
Permanganate
Hydroxylamin
e
Target
sequence is
PCR to create
heteroduplex
• Denature
• Reanneal
Treated with
respective
chemicals
and cleaved
with
piperidine
and run on

12. Single-stranded Conformation Polymorphism
(SSCP)
Conformation of Single
strand DNA is changed if
SNP is present
First denature the
double stranded DNA
and cool down on ice
Separated ssDNA
conformation by gel
electrophoresis. Result
appear in bands
SSCP enhance with
combination of
fluorescent labels and
capillary electrophoresis

14. Instruments of SSCP
Capillary electrophoresis
Amplify region of
interest by PCR
using fluorescent
labeled primer.
DNA Isolation
kit

15. MutS Protein-binding Assays
• Are the key element in
mismatch repair
• It attach with DNA and
activate MutS-H endonuclease
that cut & repair DNA
MutS
Protein
• MutS immobile in magnetic
beads to capture hetroduplex
DNA
• DNA is labeled by Biotin and
detected by ELISA
MutS protein-
Binding Assay

16. Steps of MutS protein-Binding Assay
Prepared DNA
region with
SNP using
PCR
Amplification
Step 1
Prepare
DNA
heterodupl
ex
Step 2
Formation
of MutS-
DNA
complex
Step 3
Detection
of MutS-
DNA
Complex
Step 4

17. Other Techniques
• Mismatch Repair Detection (MRD)
• Heteroduplex Analysis (HA)
• Denaturing High Performance Liquid Chromatography (DHPLC)
• UNG-Mediated T-Sequencing
• RNA-Mediated Finger printing with MALDI MS Detection
• Sequencing by Hybridization
• Direct DNA Sequencing
• Single-feature polymorphism (SFP)
• Invader probe
• Allele-specific oligonucleotide probes

18. Cont…
• PCR-based methods
• Allele specific primers
• Sequence Polymorphism-Derived (SPD) markers
• Targeting induced local lesions in genomes
(TILLinG)
• Minisequencing primers
• Allele-specific ligation probes

19. Applications
• Agriculture
• Example of Rice, Maize, canola, barley, sugar
beet etc.
• Aquaculture
• Example of Catfish

20. Cont…
• Pharmacogenetics
• Example of CYP2C19
variants
• Research
• Example of all Above

21. Thank you