The term DNA sequencing refers to "determination of precise order of nucleotide in a fragment of DNA". These days the most important and powerful technique which is available to the molecular biologist is DNA sequencing. There are two different techniques which are developed simultaneously. One is chain termination method by F. Sanger and A. R. Coulson from UK and the second one is chemical degradation method by A. Maxum and W.Gilbert from USA. Both methods imply different procedure to sequence the several Kilo basis of DNA in minimum time. But in due course of time chain termination method also known as Sanger Coulson method achieved more popularity than Maxam Gilberd method. This was mainly because the chemicals used in Maxum Gilbert method are highly toxic causing health hazard to the user.
Requirement of Chain Termination method
The chain termination method requires single stranded DNA and M13 vector for cloning the single stranded DNA. This method also needs primer ,Radio labeled deoxynucleotide like dATP (2' deoxyadenosine 5' phosphate), dGTP(2' deoxyguanosine 5' phosphate), dTTP ( 2' deoxythymidine 5' phosphate) and dCTP (2' deoxycytidine 5' phosphate) along with a single modified nucleotide known as dideoxynucleotide e.g ddTTP (dideoxythymidine 5' phosphate), ddATP (dideoxyadenosine 5' phosphate), ddCTP(dideoxycytidine 5' phosphate) and ddGTP (dideoxyguanosine5'phosphate). The dideoxynucleotide lacks 3' and 2' hydroxy group of its sugar component. It can be added in growing polynucleotide chain with same efficacy as with normal nucleotide.
Steps of Chain termination method
The first step is to anneal a short nucleotide primer on to the M3 molecule. The primer is further extended by Klenow DNA polymerase enzyme. This enzyme starts incorporating dNTP corresponding to the base present in DNA fragment. If dideoxynucleotide gets incorporated in growing polynucleotide strand, it blocks the strand synthesis e.g : If ddCTP is added, termination occurs at position opposite to Guanine base in the template. But it is to be noted that termination doesn't always occur at first Guanine base because normal dCTP is also present. So, the ratio of dCTP and ddCTP is such that an individual strand can be synthesized for a considerable distance before a ddCTP molecule is incorporated.
Procedure of Chain termination method
The strand synthesis reaction is carried out four times in parallel which includes the reaction with ddATP, ddCTP, ddGTP, ddTTP. The result is four distinct families of newly synthesized polynucleotide one family consisting of strand all ending with ddCTP, other family consisting of strands all ending with ddATP and so on.
In next step polyacrylamide gel electrophoresis is done to separate the component of each family so that length of each strand can be obtained. A very thin polyacrylamide gel which contains urea is utilized at high voltage. Urea denatures the DNA so that the newly synthesized strands get separated from the template. High voltage heats up the gel up to 60 degree centigrade which confers that two strands do not re-associate. Autoradiography is used for visualization of DNA bands as dNTP are radio labeled.
Determination of DNA sequence from Autoradiography
The band which is located at the bottom of the gel, represents the smallest piece of DNA. This strand is terminated because of the incorporation of ddNTP at the first position in the template. The track in which this band occurs is noted. Let us consider it is track of Guanine, the first nucleotide in the sequence will be G. The next most mobile band corresponds to the DNA molecule which is one nucleotide longer than the first. The track is noted and same process is followed until the largest band can be tracked. It is possible to read a sequence of about 4000 nucleotide from one autoradiograpgh e.g if the autoradiography shows 5' AGTTAGCCTA 3' sequence then the sequence of DNA will be 5' TAGGCTAACT 3'.
Significance of Chain Termination method
It is a widely used method to sequence a DNA which is usually 800 to 1000 nucleotide long.
The reason for fame of Chain termination method among molecular biologist is the relative ace with which this technique can be automated. This is important in modern research because it is not possible to carry out large number of sequencing experiments without automation.
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