This document discusses single nucleotide polymorphisms (SNPs). It defines SNPs as variations in DNA sequences that occur when a single nucleotide differs between members of a species. SNPs are the most common type of genetic variation. The document outlines the characteristics of SNPs, how they are used as genetic markers, and various methods for SNP genotyping, including direct sequencing, TaqMan assays, and microchips. It also discusses the advantages and applications of SNPs in areas like gene discovery, disease risk profiling, and genetic variation studies.

1. ASSIGNMENT ON
SINGLE NUCLEOTIDE POLYMORPHISM (SNP)
CHANDAN
HALDAR
FBT MA4- 03

2. INTRODUCTION
 Polymorphism is a generic term that means
'many shapes‘. It is the ability to appear in
different form .
 A single nucleotide polymorphism (SNP) is a
DNA sequence variation occurring when a
single nucleotide - A, T, C, or G - in the
genome differs between members of a
species (or between paired chromosomes in
an individual).

3.  Single nucleotide polymorphisms or SNP, are the
most common type of genetic variation among
species.
 Each SNP represents a difference in a single DNA
building block, called a nucleotide .
 For a variation to be considered a SNP, it must
occur in at least 1% of the population.
 For example, two sequenced DNA fragments from
different individuals, AAGCCTA to AAGCTTA,
contain a difference in a single nucleotide .

5. CHARACTERISTICS OF SNP
 In human beings, 99.9 percent bases are
same.
 Remaining 0.1 percent makes a person
unique.
Different attributes / characteristics / traits
How a person looks, diseases he or she
develops.
 These variations can be:
Harmless (change in phenotype)
Harmful (diabetes, cancer, heart disease,
and hemophilia).

6. SNP AS GENETIC MARKERS
 SNPs are found in
 coding and (mostly) non coding regions.
 Occur with a very high frequency
 about 1 in 1000 bases to 1 in 100 to 300 bases.
 The abundance of SNPs and the ease with which they
can be measured make these genetic variations
significant.
 SNPs close to particular gene acts as a marker for that
gene.
 SNPs in coding regions may alter the protein structure
made by that coding region.

7. TYPES OF SNP
SNPSNP
NON-
SYNONYMOUS
MISSENSE NONSENSE
SYNONYMOUS
In the coding
region

8. DEVELOPMENT OF SNP ( DIRECT SEQUENCING
METHOD)
Rohu
A B C (individuals)
xa,ya,za xb,yb,zb xc,yc,zc.
(genes)

9. RNA is taken from each individual.( particular
gene)
cDNA
PCR
Electrophoresis of each PCR product.
Gel extraction
Sequencing (Sanger di-deoxy method.)

10. SANGER DI-DEOXY METHOD

12. ALIGNMENT OF THE SEQUENCE
 For alignment of the genes a bioinfomatics
tool
BLAST (Basic Local Alignment Search Tool)
is used.
 Xa = AAGCCTAGATAGCT.
 XB = AAGCTTAGATAGCT.
 XC = AAGCATAGATAGCT.

13. NEXT GENERATION SEQUENCING
1)Polony sequencing
2)454 pyrosequencing
3)Illumina (Solexa) sequencing
4)SOLiD sequencing
5)Ion Torrent semiconductor sequencing

14. Micro-reactors
Adapter carrying
library DNA
Anneal DNA template
to capture beads
Break micro-reactors
Isolate DNA containing
beads
“Water-in-oil”
emulsion
+ PCR Reagents
+ Emulsion Oil
Perform emulsion PCR
A
B
Single test tube generation of millions of clonally amplified
sequencing templates
No cloning and colony picking

18. SNP GENOTYPING
 •Reverse Dot Blots
 •Direct Sequencing
 •HPLC Genotyping
 •TaqMan Assay
 •Fluorescence Polarization (FP)
 •Mass Spec
 •Microchips
 •Pyrosequencing
 •Allele Specific Hybridization
 •SNaPshot

19. Taq Man Assay
•Uses a probe that binds to center of DNA
sequence you are amplifying
•Probe contains both:
–Reporter dye
–Quencher dye –stops reporter from
fluorescence
•Whenever two dyes break apart ,we see
fluorescence of reporter dye
•Probe is allele specific so that it will only
break upon copying exact DNA sequence

21. MICROCHIPS
 Silicon chip has thousands of oligo probes
attached to it.
-Exact position of each probe is known
-Probes are allele specific
 PCR products are labeled with fluorescent
dyes.
 Washed over microchips.
 Product will bind to specific probe based on
complimentary base pairing –Position of label
will show which probe bound.

24. VALIDATION OF SNP BY Tm SHIFT PRIMER
short GC tail. (Allele1)
long GC tail. (Allele 2)

25. ADVANTAGES & DIS-ADVANTAGES OF SNP
 It is a co- dominant marker.
 PCR products can be very small:–Markers
will work with extremely degraded DNA
samples.
 More common in genome.
 Sample processing may be completely
automated.
Disadvantages:
 Its PIC (polymorphic information content)
is lower than microsatellite markers because,

26. APPLICATION
 Genetic variation between the members of
same species.
 Gene discovery and mapping.
 Diagnostics/risk profiling.
 Response prediction.
 Homogeneity testing/study design.
 Gene function identification.

27. REFERENCES
 Aquaculture Genome Technology- Zhanjiang
(John) Liu.
 Next Generation Sequencing and Whole
Genome Selection in Aquaculture- Zhanjiang
(John) Liu
 www.wikipedia.com