Sanger

sequencing Maxam–Gilbert sequencing method

method

Frederick Sanger
 Who discovered Allan Maxam and Walter
and
and at what year  Gilbert in 1976–1977.
colleagues in 1977,

1. No premature termination due to DNA

sequencing. So, no problem with polymerase to
1.high accuracy synthesize DNA
2. suitable for small 2. Purified DNA can be read directly.
3 Advantages
projects
3. can be used to analyzed nucleic acid structure
3. Easy to analyzed and epigenetic modification to DNA

1. Scalable to few
genes only 1.time consuming
2. low through out 2.as the size of DNA increase lager gels are
3 disadvantages  put required for the separation such large gels are
difficult to handle
3. large amount of
start up materials 3. use of toxic radioactive chemicals are hazardous
are required

Reagents used   A DNA dimethyl sulfate and hydrazine

polymerase
enzyme.
 A primer,
which is a
short piece of
single-
stranded
DNA that
binds to the
template
DNA and
acts as a
"starter" for
the
polymerase.
 The four
DNA
nucleotides
 (dATP,
dTTP, dCTP,
dGTP)
 The template
DNA to be
sequenc

This is the process

of selective
incorporation of This is the method of DNA sequence based on the
chain terminating nucleotide specific partial chemical modification
Description 
dideoxynucleotide and subsequent DNA cleavage.
by DNA polymerase
during in vitro DNA
replication

This method
generally is an In-
Purification of the DNA fragment that to be
Vitro synthesis of
sequenced and labeled with radioactive material.
DNA strand and by
Chemical treatment generates breaks at a specific
using terminators
nitrogenous bases and thus a series of labelled
(di-
fragments is generated. The fragments in the four
deoxynucleotide)
reactions are arranged side by side in gel
the growing strand
Principle  electrophoresis for size separation. The fragments
terminates at
visualize in X-ray for autoradiography. To visualize
specific site. Upon
the fragments, the gel is exposed to X-ray film for
termination the
autoradiography, yielding a series of dark bands
strands are overlap
each corresponding to a radiolabeled DNA
to get original
fragment, from which the sequence may be
sequence of
inferred.
unknown DNA
Strand.

Sanger sequencing
method is the chain
termination method
Maxam–Gilbert sequencing method is the chemical
that uses less
method of sequence that uses large amount of
hazardous
chemicals including radioactive material and
Major difference  chemicals and less
hydrazine and it is more sensitive and specific than
sensitive and less
Sanger sequencing method.
specific than
Maxam–Gilbert
sequencing
method.