Biotechnology Forums

Full Version: Single Nucleotide Polymorphisms
You're currently viewing a stripped down version of our content. View the full version with proper formatting.
Polymorphisms, in simple words can be described as variations. Polymorphisms which occur with a single nucleotide are SINGLE NUCLEOTIDE POLYMORPHISMS. Significance of SNPs are based on various factors like location, frequency, functional contribution to an organism.
SNPs could contribute to shortening of polypeptides, where the byproduct of the SNP could be a stop codon. These incomplete polypeptide becomes nonfunctional. SNPs that change the amino acid and therefore the destiny of the protein itself are the ones which gain more public interest. Some SNPs changes the amino acid in the polypeptide but still the protein would be as functional as a normal one these types are called silent SNPs, which normally do not have any significance. SNPs gain much of attention because of its critical role in a disease, onset of a disease (risk factor), drug response- hence pharmacogenomics, molecular marker etc.
Analyses of SNPs involve a variety of techniques. Various techniques can be used in identifying SNP like;
i. Real-time PCR
ii. Microarray
iii. Mass spectrometry
iv. Restriction Fragment Length Polymorphism

One of its application is identifying the risk factor for a disease and evaluating SNP significance in a population. It is explained by an example below: Disease: Hypertension, Polymorphism: Apolipoprotein E. Apolipoprotein E (Apo E) is a 299-amino acid polypeptide synthesized in the liver and intestine. The gene that provides the instructions for making Apo E is called APO E gene. It is located in the long arm of chromosome 19 in a cluster with APOC1 and APOC2. The APO E gene locus is polymorphic with three major known alleles; APO E3, APO E4 and APO E2. (http://ghr.nlm.nih.gov/gene=apoe). APO E3 is the common allele present in most populations with the highest frequency. Investigations on Apo E gene and hypertension has suggested a protective role of APO E2 where, APO E4 is thought to increase the risk of high blood pressure.

RFLP method of identification involves ; DNA extraction from a control set as well as Known set for the disease. PCR Amplification of sequence using specific primers for APO E. Restriction Fragment Length Polymorphism- Selecting appropriate restriction enzyme is of utmost importance. Restriction Enzyme ; HhaI. PCR-RFLP analysis of the HhaI polymorphism at the APOE gene locus shows six possible genotypes based on the presence/absence of the restriction site. These genotypes are E3E3, E2E3, E3E4, E2E2, E2E4 and E4E4.Gel Electrophoresis- The presence or absence of the restriction site could give differences in the band size depending upon the genotype. The band sizes are 91bp, 81bp, 72bp, and 48bp

Analysis of SNP can be done by statistical evaluation of the result obtained in a population for the genotype frequency, frequency of allele in the population, its significance in diseased and control set. Calculating Genotype Frequency: using simple gene-counting method in determining the genotype frequency of Apo E genes. Calculating Allele Frequency the same way of calculating genotype frequency. Significance study using Hardy-Weinberg Equilibrium either using softwares (eg. GENEPOP) or manually.

After the identification of the SNP, the SNP can either be uploaded on the database for SNP (dbSNP) or JSNP. Curated SNPs have Reference SNP number.