DNA sequencingThe dideoxy approach

Rajat Patil – 2bv13bt037Praveenkumar Tagare – 2bv13bt032

IntroductionMuch of the progress in recent molecular biology and genetic engineering is due to the ability to sequence the DNA. Although large scale efforts of this sort like the Human Genome Project (HGP) used more advanced approaches, the basic DNA sequencing capability arouse from the dideoxy method of DNA sequencing. This method is therefore foundational for most of the research in the field.It can be applied for short sequences only. Large sequences need to be fragmentized.

• To understand the dideoxy method of DNA sequencing, it is important to know the basic structure of DNA and the nucleotides.• Its made up of three components,• Sugar base – deoxy ribose• Phosphate group• Nitrogenous base ( A, T, G or C)

• Accordingly they are called dNTP’s. (dATP, dGTP, dCTP, dTTP)

• These lack the hydroxyl group at 3’ carbon atom on the sugar.

Deoxy nucleotides

Dideoxy nucleotides• Its also made up of three components,• Sugar base – Dideoxy ribose• Phosphate group• Nitrogenous base ( A, T, G or C)

• Accordingly they are called ddNTP’s. (ddATP, ddGTP, ddCTP, ddTTP)

• These lack the hydroxyl group at both 3’ and 4’ carbon atoms on the sugar.

• In a DNA molecule, each dNTP is bound to the consequent dNTP by a phospho diester bond between 4’ carbon atom of one dNTP and the phosphate group of the next dNTP.

• But this is not the case for ddNTPs since they lack the hydroxyl group at 4’ carbon atom of the sugar.

• So a chain of DNA terminates when a ddNTP is present.

• Using this property of a ddNTP, we can sequence a DNA.• Consider a short segment of DNA to be sequenced, - T G A

T T C• Four copies of the same sequence are prepared.• It is fed to the PCR and all the necessary enzymes and other chemicals

are added.• To each of the copy, three dNTPs are added and one ddNTP is added in

equal volumes• 1 - dATP, dGTP, dCTP and (dTTP : ddTTP = 1:1)• 2 - dATP, dTTP, dCTP and (dGTP: ddGTP = 1:1)• 3 - dATP, dTTP, dGTP and (dCTP: ddCTP = 1:1)• 4 - dCTP, dTTP, dGTP and (dATP: ddATP = 1:1)

• The four different samples may give rise to many combinations of different sized DNA fragments upon PCR.

• Now, load these fragments onto a gel and carry out electrophoresis to separate the fragments based on their size.

• After a while, the bands are obtained. The lighter DNA segments travel furthest and the heavier DNA segments travel the least.

• To determine the sequence of DNA, we move back from the lightest DNA segment which is a single dideoxy nucleotide.• This chain terminated at the initial point indicating the

sequence starts with the dNTP of that ddNTP.

• Similarly, the next farthest band, which will be a di-nucleotide and the second nucleotide is the dNTP of that ddNTP in which the band was observed.

• Similarly, all the bands are analyzed and a sequence is created. We cannot predict the last nucleotide because it is not known whether termination occurred because of the ddNTP or not.

CONCLUSION• This was the first ever method to sequence DNA.• The longer the sequence, the lengthier is the gel required.• Its limitations are overcome by using shotgun sequencing and

hierarchal methods for sequencing of the very large genomes of eukaryotes.

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