| Restriction fragment length polymorphism (RFLP) : Restriction fragment length polymorphism (RFLP) is the simplest and easiest method of SNP detection. RFLP uses a huge number of restriction endonucleases which have affinity for specific restriction sites. Firstly, the genome sample is digested by these restriction endonuclease and the length of frangments is determined by gel assay to determine whether a particular enzyme digests a specified restriction site or not. Thus a failure in cutting the genome sample at expected restriction site indicates that there is a mutation at the point of the restriction site which is rendering it protected from nuclease activity. There are several drawbacks of this technique as well, such as slow nature of gel assays make RFLP, complexity of most eukaryotic genomes, the requirement for specific endonucleases and the uncertaininty of exact mutation to identify in single experiment.
DNA sequencing: The region of the interest is sequenced using Sanger's Dideoxy DNA sequencing method. Then the sequence is read to find out any variation or SNP. We have already studied method for DNA sequencing in our earlier lectures.
Taq-Man Assay: It uses a probe which binds to the SNPs as it is specific. TaqMan assay for SNP genotyping uses Taq DNA polymerase's 5'-nuclease activity. The TaqMan assay is performed concurrently with a PCR reaction and the results can be read in real-time as the PCR reaction proceeds. Since the TaqMan assay is based on PCR it is simple and easy and can detect upto seven SNPs in one reaction. However, each SNP requires different probe for identification, therefore it cannot detect closely assembled SNPs the genome.
Mass spectrometry: In this method four different ddNTPs are used i.e. four different molecular weights. Mass spectroscopy is done after running the PCR to find out the exact weight of the product thus SNP can be detected. There are some other techniques available for detection of SNPs as well, such as Molecular Beacon, Hybridization analysis etc.
Applications of SNPs
There are several applications of SNPs in disease diagnosis and other aspects as follows
Use of SNPs in disease diagnosis: The genome of every individual has unique SNP pattern made up of different genetic variations. Although, most of these SNPs are housekeeping and does not cause any type of disease, yet they may serve as biological markers for identification of a particular disease as they are commonly occurring near to a gene associated with a disease. Occasionally, SNPs may also be responsible for a disease cause due to genetic variation. Therefore, scientists are actively involved in identification of genetic make up and SNP pattern of individuals affected with any disease and prepare a database for further use as reference.
Use of SNPs in public databases: SNPs are present throughout the genome, thus they serve as biological marker i.e. have identifiable physical location that can be easily tracked and used for constructing a chromosome map and can show a position of a gene. NCBI has prepared a huge public SNP database (dbSNP) for comparision of genome of several species. This database can be assessed by any research community to compare and identify the genome sequence of species. |
