How Sanger Sequencing Works? (Classic Sanger Method)

what do we mean by DNA sequencing it is a technique by which the precise order of nucleotides in a DNA segment can be determined today we are going to discuss original Sanger method of DNA sequencing this method forms the basis of modern computer automated sequencing techniques Sanger sequencing was developed by Frederick Sanger and colleagues at the University of Cambridge in 1977 this technique involves in vitro DNA synthesis it is based on the principle and biochemistry of DNA replication tell me what will be the basic requirements for in vitro DNA synthesis first we should have a DNA template strand this strand will provide complement three bases for the synthesis of the new strand here this is 3 prime end of template strand and this is 5 prime end next we require a primer as you know a primer is a short oligonucleotide sequence complementary to the sequence at the three prime end of the template strand it serves as the starting point for new strand synthesis we will also require the auxin nucleotides to catalyze the DNA synthesis reaction we require DNA polymerase DNA polymerase incorporates deoxynucleotides complementary to the template strand and extends the DNA chain in five prime to three prime direction now here we need to note very important point primer is one of the essential requirements of DNA synthesis but why because DNA polymerase cannot catalyze the synthesis reaction on its own it requires three prime hydroxyl group to form phosphodiester bond with the incoming deoxynucleotide and this initial three prime hydroxyl group is provided by the primer once DNA synthesis is initiated each deoxynucleotide incorporated in the growing DNA chain has a hydroxyl group of the 3 prime position of the deoxyribose molecule and thus DNA polymerase keeps on elongating the chain sänger DNA sequencing technique makes use of modified deoxynucleotides known as DD ox and nucleotides now what are these videos and nucleotides and what is their function in DNA sequencing look at this image this is a basic chemical structure of a dioxin nucleotide here you can see that at the three prime position of sugar hydroxyl group is present this is the three prime hydroxyl group which participates in phosphodiester bond formation during DNA synthesis now look at this image here the hydroxyl group is absent at the three prime position of the sugar instead there is a 3 prime hydrogen this is d deoxynucleotide if in a DNA synthesis reaction Ana deoxynucleotide is added the DNA synthesis will terminate with the incorporation of this D deoxynucleotide this is because now there is no three prime hydroxyl group for the further extension of DNA chain for this reason the dioxin nucleotides are called chain termination nucleotides and Sanger technique is also known as chain termination method or D deoxy DNA sequencing so if the concept of de deoxynucleotide is clear to you it will be easy to understand the procedure and interpret the results of Sanger sequencing technique let's now move on to the Sanger sequencing methis Aang a sequencing consists of four separate reactions that run parallel II we will label them as 1 2 3 & 4 each of these reactions has some components that are common to all these common components our first component is DNA template strand many copies of this strand are used in each reaction let's say this is our template strand second component is DNA primer again many copies of DNA primer are used Sanger radio labeled these primers for detection purpose third component is DNA polymerase and fourth component consists of all four standard deoxy nucleotides or dntps they are used in large amounts besides these common components small amounts of one dideoxy nucleotide or dd ntp is added in each reaction this is the component which is different in each reaction so for our illustration let's say D D ATP is added in the first reaction D dctp and second D dgtp in third and DD TTP in fourth now as we know D deoxynucleotides or D dntps when incorporated in an elongating DNA chain they will terminate DNA synthesis reaction since small amounts of DD ntp are included in each reaction DNA synthesis will not terminate every time but DNA polymerase will occasionally insert a DD ntp instead of a dntp into a growing DNA strand since the incorporated DD ntp lacks 3 prime hydroxyl group it cannot form a bond with another nucleotide and DNA synthesis will terminate thus in each case we will get partially replicated fragments let's understand this for the first reaction this is our DNA template strand and this is the primer deedy ATP is present in lower amount DD ATP will be incorporated occasionally wherever normal dat P is to be incorporated complementary to thymine residue and each time a DD ATP has incorporated the DNA synthesis terminates so as you can see there are three thymine residues in the template strand complimentary to these thymine residues DD ATP can be incorporated in the new strand at the end of this reaction there will be three partially replicated DNA fragments similarly three partial replication products will be produced in second reaction where DD CTP will be incorporated occasionally complementary to guanine residue in the third reaction to partial replication products are produced and in the fourth reaction for partial replication products are produced upon the complete of the four parallel reactions partial replication DNA products will occur for every nucleotide in the template that means each reaction has now a set of DNA fragments that have same starting point but different end points in the next step these reaction mixtures are subjected to polyacrylamide gel electrophoresis the contents of each reaction are loaded into separate lanes of a DNA electrophoresis gel polyacrylamide gel electrophoresis separates the partial DNA fragments of each reaction on the basis of their size smallest fragments migrate to maximum distance in the gel and largest fragment migrates to the minimum distance next step is determination of sequence of the DNA segment since Sanger radio labeled the primers in each reaction mixture autoradiograph of the gel was obtained Auto radiograph is a photograph of gel produced by radiation from radioactive material present in the partial DNA fragments in the gel let's say this is the auto radiograph of the gel we will label each lane according to the dntp with which the partial DNA fragments should end now let's understand the interpretation of DNA sequence as we know bands are arranged according to their size smallest fragment is found at the bottom of the auto radiograph and largest fragment is found at the top we will read the sequence from bottom to top according to their increasing size shortest fragment is in the a line at the bottom this shortest fragment means that the dideoxy ATP was added nearest to the three prime end of the primer the second shortest fragment is in the T Lane then in the G Lane then in the see Lane and so on thus the sequence which we read in this Auto radiograph from bottom to top is a T G see a see T T G a T see the direction is 5 prime to 3 Prime from bottom to top now the sequence we read is the sequence of the newly synthesized strand so what will be the sequence of template strand it will be complementary to the sequence read from autoradiograph so this is five prime end of the template strand and this is three prime end and the sequence is G a t see a a g t G see a T thus we successfully obtained the sequence of given DNA strand that's all in today's video lecture I hope it is helpful to you thank you for watching

Channel: Frank Lectures

Views: 122,536

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Keywords: sanger sequencing method, sanger sequencing animation, sanger sequencing, principle of sanger sequencing, dideoxy sequencing, chain termination method

Id: -QIMkQ4E_wE

Length: 11min 55sec (715 seconds)

Published: Sat May 25 2019