hey guys with my chemistry basics here let's talk about Sanger sequencing method now before moving further the Sreenu is divided into three parts the first part explains the principle of the method the second part is about classical Sanger sequencing that was used widely in 1980s and the third part is about modern Sanger sequencing that uses fluorescent dyes Sanger sequencing is based on dye deoxy nucleotides in DNA polymerizing reaction let's understand what are dye dioxin nucleotides the nucleotide has three components the nitrogenous base which can either be a T G or see the sugar residue and
the phosphate attached with the sugar residue when all three components are present it's called nucleotide if the phosphate is absent then it's called nucleoside now watch carefully if the hydroxyl group at the two - end of the sugars removed then it's called deoxynucleotide the deoxynucleotides are present in the DNA and they are used as a substrate by the enzyme DNA polymerase all the DNA polymerase whether it's DNA polymerase 1 2 3 tack polymerase etc uses dntps as their substrate the hydroxyl group attached at the 3 - end plays a very important role and DNA polymerizing
reaction during DNA polymerization there is a reaction between 3 - o h group and the phosphate of the new DNT be in 1977 Frederick Sanger had a brilliant idea if the three - Oh H group of the nucleotide is absent then there won't be addition of new nucleotides and the reaction would stop the nucleotide having three - hydroxyl group absent is call die deoxy nucleotide die means - and deoxy means absence of the hydroxyl group let's see classical Sanger sequencing method the template DNA to be sequence is divided into four tubes each tube has a
primer that will bind to the template DNA all the four dntps that is datp dctp dgtp and dctp are present along with these DNA polymerase enzyme is present that will add dnt B's on the template DNA now your comes a very interesting part this reaction mixture also has one radio label die deoxy nucleotide at a very low concentration what I mean by this is that tube one will have died deoxy ATP tube two will have died deoxy TTB tube three will have died deoxy gtp and tube for will have died the oxy CTP at a
very low concentration and all of them will be radio labeled now let's see what happens in tube one the DNA polymerase would add dntps on the template DNA and carry out normal polymerization reaction if somehow the dye deoxynucleotide is incorporated then the reaction suddenly stops the reaction terminates as there is no three - hydroxyl group for the addition of new dntps the incorporation of dye deoxy nucleotide and termination of polymerizing reaction is a random process hence the overall mixture will have several fragments that has experience chain termination now if the length of the fragment is
known then the location of adenine in the DNA can be estimated the length of the fragments can be obtained by performing polyacrylamide gel electrophoresis and autoradiography once the autoradiogram is obtained we can now have the sequence information of the desired DNA fragment okay the first nucleotide is going to be a the second nucleotide is a third is G fourth is C fifth is T 6 is T seventh is a 8 is g9c and 10th is again si now let's see Morgan Sanger sequencing method which is used even today now in this method each of the
dye deoxynucleotide is attached with a fluorescent dye the fluorescence dye can either be yellow green blue or red instead of having an extra film placed on a gel will now have a gel that would give fluorescence when exposed to UV light as each of the guy deoxynucleotide have their own fluorescence it's now possible to carry out the reaction in one single tube rather than four different tube the chain termination reaction would result if dye deoxynucleotide is incorporated in the DNA the chain terminated fluorescence fragments can be separated by polyacrylamide gel electrophoresis in a single well
the more advanced system uses capillary electrophoresis for the separation of fluorescence DNA fragments these capillaries are further equipped with CCD or charge-coupled device for the detection of fluorescent signals [Music]
