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