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Sanger's Sequencing

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9 views9 pages

Sanger's Sequencing

Uploaded by

baneenfatima705
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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SANGER’S

SEQUENCING
Khadijah Asif
Saima Farooq
DEFINITION

 Sanger sequencing, also known as the “chain


termination method”, is a method for
determining the nucleotide sequence of DNA.
Sanger sequencing requires a DNA template,
a sequencing primer, a thermostable DNA
polymerase, nucleotides (dNTPs),
dideoxynucleotides (ddNTPs), and buffer.
METHOD
 The DNA sample is heated to form ssDNA and
divided into 4 tubes. Each tube will have a
primer that will bind to template DNA. All
dNTPs along with DNA polymerase are present.
Polymerase adds the dNTPs on the template.
 The reaction also has one radiolabeled ddNTP
at very low concentration.
 The mixture is first heated to denature the
template DNA (separate the strands), then
cooled so that the primer can bind to the
single-stranded template. Once the primer has
bound, the temperature is raised again,
allowing DNA polymerase to synthesize new
DNA starting from the primer. DNA polymerase
will continue adding nucleotides to the chain
until it happens to add a dideoxy nucleotide
instead of a normal one. At that point, no
further nucleotides can be added, so the
strand will end with the dideoxy nucleotide.
 the tube will contain fragments
of different lengths, ending at
each of the nucleotide positions
in the original DNA.
 Length of the DNA Is estimated
by Gel Electrophoresis.
 The DNA is loaded on gel matrix
and electric current is applied.
 Because all fragments has same
charge per unit of mass so the
speed at which the
oligonucleotide move will only be
determined by size.
 The small fragments move faster
while the longer one will move
slower.
Uses of Sanger DNA Sequencing
in Genetic Engineering
1-CLONING
WHY DO E NEED CLONING..?
1. Production of recombinant human insulin.
2. In identification of human diseases.
3. To produce live recombinant vaccines.
4. CRISPR-mediated genome editing with Sanger
sequencing.
5. HLA typing.
6. To determine the sequences of many relatively
small fragments of human DNA.
7. Sanger sequencing solutions for SARS-CoV-2
research.
LIMITATIONS
1. Inability to detect large deletions or
duplications of sequences
2. Can only sequence short DNA fragments of
about 300 to 1000 base pairs.
3. Sequence quality degrades after 700 to 900
bases
4. Not suitable for parallel testing.
DUE TO ALL THESE LIMITATIONS IT
FAILED

 But Wait …….


IT IS STILL USEFUL WHEN ….
Sequencing single genes
Sequencing amplicon
targets up to 100 base
pairs.
sequencing 96 samples
or less.
 analyzing short
tandem repeats
(STRs).

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