DNA sequencing methods.pdf
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Wilson.G.Anandaraj
I M.Sc Environmental Biotechnology
Bharadhidasan University.
FOR CONTACT
:
anandaraj.wilson@gmail.com
WHAT IS DNA SEQUENCING ?
DNA sequencing usually involves enzymatic DNA
synthesis in the presence of base-specific
dideoxynucleotide chain terminators
.
dideoxynucleotide chain terminators
.
Determining the DNA sequence is therefore useful in
basic research studying fundamental biological
processes, as well as in applied fields such as
diagnostic or forensic research
.
FOUNDERS OF SEQUENCING TECHNOLOGY
Sanger
Wally Gilbert
MAXAM & GILBERT DNA SEQUENCING
(CHEMICAL DEGRADATION)
In the late 1970s, A. M. Maxam and W.Gilbert
devised the first method for sequencing DNA
fragments containing up to
500 nucleotides
.
the sequence of a double-stranded DNA molecule is
determined by treatment with chemicals that cut the
molecule at specific nucleotide positions
.
molecule at specific nucleotide positions
.
it is the early method involving base-specific chemical
modification and subsequent cleavage of DNA
.
most of the chemicals used in chemical degradation
method are toxic and hazardous to the health of the
researchers doing the DNA sequencing
.
CHEMICAL DEGRADATION METHOD
PROCEDURE
four samples of an end-labeled DNA restriction fragment are
chemically cleaved at different specific nucleotides
.
the resulting sub-fragments are separated by agarose
gel electrophoresis and the labeled fragments are
detected by autoradiograph.
the sequence of the original end-labeled restriction fragment
can be determined directly from parallel electrophoretograms
of the four samples.
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