Known SNP genotyping.

This is one of the highly studied methods in the biology field. After the invention of PCR by Mullis a dynamic changes has occurred in this field. Broadly we can classify this in to 2 ,one is conventional PCR RFLP and the other one is multiplexing.

PCR RFLP: In this we will amplify a short segment of DNA which contains our SNP and will cut the amplified DNA with help of an enzyme. These enzymes are known as restriction enzymes. Restriction enzymes are nothing but DNA cutters which cuts DNA at a particular sequence for example EcoR I enzymes the DNA which is having the sequence G/AATTC (/ indicates the site of cut). In this case if we want to see whether GAATTC the A in red is mutated or not. We can need to amplify 100-200 basepairs DNA around this particular sequence and the after amplification we have to incubate the amplicon along with the enzyme. After that we need to run the digested product on to a gel and then we have to see whether the enzyme cut the DNA or not. If the enzyme cuts the DNA means there is no mutation at that particular base and the restriction site is preserved. If there is any mutation the enzyme will not cut the amplified DNA. This method is old fashioned today because it consumes much time when compared to the other applications available. Another restriction is we may not get the enzymes for all the sites which we want to screen.

TaqMan. This application is very rapid and accurate. This method works on the principle that the Taq polymerase have 5’ exonuclease activity. In this we will have a primerset and a probe which is labelled with a Vic and florescent Dye. A Vic blocks the Dyes florescent activity until it is with the probe. If the probe get digested the Vic and Dye will be separated then florescence of the dye will be detected by the system.

In SNP detection we will design a probe in such a condition which will be specific for our SNP and a primer set with the specificity of our (ROI)
this will be further explained in future posts be in touch

Methods to detect SNPs

There are several methods to SNP identification.
SNP identification can be categorized in to 2 main sub sets.
1. Novel SNP detection.
2. Simply detecting the known SNPs.

For novel snp detection sequencing will be the best method. In this we will select the region which we want to screen for snps from a public database like Ensembl and/or NCBI and we will sequence the region using primers which are specific for that particular region. Once we sequence the region of interest (ROI) we have to compare the sequence to the preexisting sequence to see the variations(SNPs). Here we will get a doubt if we don't have a previous sequence knowledge of that ROI the how can we identify the SNPs. In such case if you are the first person to perform the sequence analysis for that ROI then you have to do the same thing with some other samples after that compare all the sequence to each other and then you will come to know whether there is any variation present in that ROI. To do this we have to use sequence alignment softwares. These softwares align sequences to each others, by doing this we can compare them very fast and very easily. Once we get an idea about the SNp sites in our ROI the we can go for known SNP detection.
Known SNP detection is also known as SNP genotyping. There are several methods available for this. We will discuss about them in future.

If you have any observations please don't hesitate to contact us. Be in touch........

Single Nucleotide Polymorphism(SNP)

A single nucleotide polymorphism, or SNP (pronounced snip), is a DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles : C and T. Almost all common SNPs have only two alleles.

Within a population, SNPs can be assigned a minor allele frequency - the ratio of chromosomes in the population carrying the less common variant to those with the more common variant. It is important to note that there are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another. In the past, single nucleotide polymorphisms with a minor allele frequency of greater than or equal to 1% (or 0.5%, etc.) were given the title "SNP," an unwieldy definition. With the advent of modern bioinformatics and a better understanding of evolution, this definition is no longer necessary.

DNA strand 1 differs from DNA strand 2 at a single base-pair location (a C/T polymorphism).

Single nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed synonymous (sometimes called a silent mutation) - if a different polypeptide sequence is produced they are non-synonymous. SNPs that are not in protein-coding regions may still have consequences for gene splicing, transcription factor binding, or the sequence of non-coding RNA.

Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease).

The study of single nucleotide polymorphisms is also important in crop and livestock breeding programs (see genotyping). See SNP genotyping for details on the various methods used to identify SNPs.