DNA Sequencing Services

What is DNA Sequencing?

– Determining the precise order of nucleotides in a piece of DNA.
– DNA sequence is useful in studying fundamental biological processes and in applied fields such as diagnostic or forensic research.
– DNA sequencing methods have been around for 40 years, and since the mid-1970s.

DNA Sequencing Goal: Find the complete sequence of A, C, G, T’s in DNA
DNA Sequencing Challenge: There is no machine that takes long DNA as an input, and gives the complete sequence as output

Sequencing Methods

Two basic methods for DNA sequencing :-
A- Chemical Cleavage Method
– Base-specific cleavage of DNA by certain chemicals
– Four different chemicals, one for each base
– A set of DNA fragments of different sizes
– DNA fragments contain up to 500 nucleotides
– Enzymatic method

The Sanger DNA Sequencing Method

– Uses dideoxy nucleotides to terminate DNA synthesis.
– DNA synthesis reactions in four separate tubes.
– Radioactive dATP is also included in all the tubes so the DNA products will be radioactive.
– Yielding a series of DNA fragments whose sizes can be measured by electrophoresis.
– The last base in each of these fragments is known.

Automated DNA Sequencing

– The primer extension reactions are run in the same way as in the manual method
– Reaction carried out in one tube and all possible products are actually produced
– The various reaction products separate according to size on gel electrophoresis
– The bands are color-coded according to the termination reaction that produced them
– A laser scanner excites the fluorescent tag on each band as it passes by, and a detector analyzes the color of the resulting emitted light
– Each colored peak is a plot of the fluorescence intensity of a band as it passes through the laser beam

Methods of Sequencing

A) Sanger dideoxy (primer extension/chain-termination) method: most popular protocol for sequencing, very adaptable, scalable to large sequencing projects
B) Maxam-Gilbert chemical cleavage method: DNA is labelled and then chemically cleaved in a sequence-dependent manner. This method is not easily scaled and is rather tedious
C) Pyrosequencing: measuring chain extension by pyrophosphate monitoring