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Single Nucleotide Polymorphisms (SNPs)

Polymorphism A difference among individuals, groups, or populations. Genetic Mutation A change in the nucleotide sequence of a DNA molecule. Genetic mutations are a kind of genetic polymorphism

Different types of markers

Biochemical Serum proteins Isozyme Molecular RFLP RAPD AFLP Microsatellite SNP..

What is SNP ?

A SNP is defined as a single base change in a DNA sequence that occurs in a significant proportion (more than 1 percent) of a large population. SNPs in the genome are uniformly distributed and found throughout the genome at very high frequency

SNPs are the most simple form and most common source of genetic polymorphism in the human genome (90% of all human DNA polymorphisms). SNP (single nucleotide polymorphism) is usually alternative of two possible nucleotides at a given position. Although in principle, at each position of a sequence stretch, any of the four possible nucleotide bases can be present, SNPs are usually biallelic in practice. Indels are also considered and studied under SNPs

SNP- A to G (Transition)

Sequence Variation and SNP Distribution

Sequence variation (quantity of SNPs) can be measured in nucleotide diversity: the number of base differences between two genomes over the total number of bases compared. SNP Distribution is not uniform for any of the three categories: Over a complete genome (1/3 in coding, 2/3 in noncoding). Over all the chromosomes (fewer SNPs in sex chromosomes). Over a single chromosome (SNPs often concentrated around a specific location).

Mutation mechanisms result either in

Transitions: Transversions

Purine - Purine (AG) or Pyrimidine - Pyrimidine (CT) exchanges Purine - Pyrimidine or Pyrimidine - Purine (A C, A T, G C, G T).

Transitions are more common among observed SNPs. SNPs may also occur in regulatory regions of genes. These SNPs are capable of changing the amount or timing of a protein's production. Such SNPs are much more difficult to find and understand and gene regulation itself is not yet clearly understood

Coding Region SNPs

A SNP in a coding region may have two different effects on the resulting protein

The substitution causes no amino acid change to the protein it produces. This is also called a

silent mutation.

The substitution results in an alteration of the encoded amino acid. A missense mutation changes the protein by causing a change of codon. A nonsense mutation results in a misplaced termination codon.

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, hemophilia etc.) Latent (variations found in coding and regulatory regions, are not harmful on their own, and the change in each gene only becomes apparent under certain conditions)

SNPs are found in coding and mostly in noncoding 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.


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.

SNPs may / may not alter protein structure

First -Wild Third -mutant Second-Heterozygous

First- AA (Homozygous) Third- GG (Homozygous) Second- Heterozygous (G/A)

Double muscling in Belgian blue cattle is due to 11bp deletion in Exon 3 of GDF 8 gene Mutation in Exon 3 of Myostatin gene in Piedmontese

Important concepts related to SNPs

Phenotype The observable properties of an individual as they have developed under the combined influences of the individual's genotype and the effects of environmental factors. Genotype An exact description of the genetic constitution of an individual, with respect to a single trait or a larger set of traits. The genetic constitution of an organism as revealed by genetic or molecular analysis, i.e. the complete set of genes, both dominant and recessive, possessed by a particular cell or organism. Genotyping Genotyping is normally defined as detecting the genotypes of individual SNPs. In diploid organisms (alternative alleles of SNPs), such as humans, the linkage of particular SNP genotypes on each chromosome in a homologous pair (the haplotype) may provide additional information not available from SNP genotyping alone.

One key feature of most SNP genotyping techniques, apart from those based on direct hybridisation, is the two step separation: 1)generation of allele-specific molecular reaction products 2)separation and detection of the allele specific products for their identification.

Techniques involving the generation and separation of an allele-specific product

PCR-RFLP Single strand confirmation polymorphism (SSCP) Primer extension Oligonucleotide ligation assay (OLA) Pyrosequencing Exonuclease detection High-Throughput SNP Genotyping

Steps in SNP Genotyping

Selection of a gene Generation of template and design of PCR primers Optimization of PCR Purification of PCR products and sequencing Generation of Contig Sequence Selection of SNPs Genotyping of SNPs Screening and confirmation of SNPs


are designed by primer3 software available online is to be optimized using the primers.


Standardization of PCR

Gradient PCR for all the 8 primer pairs Temperature Mgcl2

2 3

6 7

2 3

6 7

2 3

6 7 8

PCR products with different primers

PCR products purified by column / alcohol precipitation / Enzymatic treatment PCR products sequencing Generation of contig sequences


Designing of PCR-RFLP for the selected SNPs





SNP genotyping Bands

CC 256/254 Mutant

CT 510/256 Heterozygous

TT 510 Wild

Other techniques
Single strand confirmation polymorphism Based on specificity of folding confirmation of single stranded DNA, in non denaturing conditions. Based on electrophoretic mobility caused to due to mismatch/indel Base difference in lower size (up to 300bp) can be detected

SNP Applications
Diversity analysis: The data generated by screening of SNPs by different procedures can be used in constructing Phylogenetic tree and biodiversity studies. It can also be used in correlation of phenotypic or production parameters Gene discovery and mapping

Pharmacogenomics understanding the correlation between an individual patient's genetic make-up (genotype) and their response to drug treatment Goals: Patient or population specific treatments Avoidance of adverse effects or inefficacy of drugs Drug-target design

Applications SNP proteomics

SNP related functional proteomics involve the identification of functional SNPs that modify proteins and protein active sites structure and function. By identifying the modifications generated by functional (coding) SNPs in disease related proteins, "new compounds can be designed for correcting or enhancing the effects of those mutations in the population."

Association-based candidate polymorphism testing. Diagnostics and risk profiling Prediction of response to environmental stimuli Homogeneity testing and epidemiological study design.