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Monday, July 28, 2014

DNA Sequencing - Traditional and Next gen Sequencing Methods


DNA sequencing is the process of finding out the exact order of nucleotide present in the DNA molecule. DNA sequencing has revolutionized medical and biological research. Before going into the details of DNA sequencing let us have some basics on the DNA molecule.

Central Dogma of Molecular Biology


Nucleic Acid - DNA / RNA Overview

Nucleic acids are first discovered in 1869 by Miescher. Found as a precipitate that formed when extracts from nuclei were treated with acid. Compound contained  C, N, O, and high amount of P.since it was an acid compound found in nuclei therefore named nucleic acid.

DNA / RNA are made up of nucleotides, these are the building blocks. Nucleotides composed of a Nitrogenous base, a pentose sugar and a phosphate group.


DNA has Adenine,Thymine,Guanine  and Cytosine, where as in RNA the nitrogenous bases found are Adenine,Guanine, Cytosine and Uracil.

The sugar present in DNA is deoxy ribose, where as in RNA it is D-ribose. Thats the reason DNA is called as 2-deoxy ribo nucleic acid.



Nucleotides:
A nucleotide comprises of a Pentose Sugar, Nitrogenous Base and a Phosphate Group.


The arrangement of Nucleotide polymer in DNA and RNA is as follows:



In DNA Adenine pairs with Thymine by a double bond and Guanine pairs with Cytosine by triple bonds.


DNA Sequencing: 
  • Maxam Gilbert Method of DNA sequencing
  • Chain Termination Method / Sanger's Sequencing Method
Maxam Gilbert Method of DNA sequencing






Sanger's Sequencing

Sanger's sequencing method was developed by Frederick Sanger and colleagues. This method is also know chain termination method. Sanger's sequencing method requires single stranded DNA template, Primer, Taq DNA polymerase, dNTPs and modified nucleotides namely ddNTPs (dideoxy nucleotide triphosphate). when a ddNTP is incorporated into a growing chain, the chain terminates. ddNTPs are usually flourescently / radiolabelled. By looking at the emission of light from the ddNTP sequence of the DNA can be deduced. the sequencing product will be of varying lengths which can be separated by Gel / capillary electrophoresis to read out the base sequence.




Next Gen Sequencing Methods:
  • Ion Torrent Sequencing

  •  

  • Pyrosequencing

  • SOLiD Sequencing




  • Illumina Sequencing Technology

 

References

Illumina Technical Resources
Applied Biosystem, Technical Resources
Other Internet Sources