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DNA Sequencing

The document discusses DNA sequencing including its history, methods, and applications. It describes two main methods - Sanger sequencing and Maxam-Gilbert sequencing. Sanger sequencing is still widely used and was important for the Human Genome Project. DNA sequencing is used in various fields like molecular biology, medicine, forensics, and virology.

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6 views

DNA Sequencing

The document discusses DNA sequencing including its history, methods, and applications. It describes two main methods - Sanger sequencing and Maxam-Gilbert sequencing. Sanger sequencing is still widely used and was important for the Human Genome Project. DNA sequencing is used in various fields like molecular biology, medicine, forensics, and virology.

Uploaded by

M.S.R
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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DNA sequencing

Presented by
Emaan Fatima (15906)
Group 1
Emaan Fatima
Shameen Anwar
Maryum Khan
BS MLT 5th
Riphah International University Faisalabad
Contents

• What is DNA ?
• What is DNA sequencing ?
• History of DNA sequencing
• Why we need to sequence a DNA
• Methods of DNA sequencing
What is DNA and its structure ?

• Deoxyribonucleic acid, is the double


stranded molecule that contains the
genetic code of organisms.
• It contains four bases
1. Adenine (A)
2. Thymine (T)
3. Cytosine (C )
4. Guanine (G)
DNA sequencing

• DNA sequencing is the laboratory


technique used to determine the exact
sequence of bases ( A, T, G and C ) in a
DNA molecule .
• DNA sequence information is important
to scientists investigating the functions
of genes.
History of DNA sequencing

• Very first method used for DNA sequencing were created by Ray Wu in
the 1970s a Chinese American biologist based at Cornell University.
Using highly labelled deoxynucleotides (single units of DNA) and DNA
polymerase he found a way to sequence the terminal region of a DNA
molecule.
• That time researches were only able to sequence a small number of
base pairs.
• In1977, Sanger and his colleagues announced technique called the
'Sanger method' or 'dideoxy sequencing'. This made it possible to
sequence much longer stretches of DNA very rapidly.
History cont.....

• In the same year , the chemical method of DNA sequencing was


explained by Allan Maxam and Walter Gilbert.
• By 1990 things had improved, then some laboratories able to
sequence hundred thousand bases .
• That was costly and impractical .
• Automation made the process much faster and more practical.
• Now , individual genes are sequenced on a regular basis and can
be done quickly and affordably in laboratories.
Why we need to sequence a DNA molecule ?

1. Molecular biology
It can be used to find genes , segments specific for a
protein.
2. Medicine
To confirm the identity of a clone or a mutation.
3. Forensics
Used for DNA fingerprinting
4. Virology
Sequencing is one of the main tools in virology to identify
and study the virus.
Methods for DNA sequencing

• There are two basic


methods for DNA
sequencing ;
1. Sanger sequencing or
chain termination method
2. Maxam-Gilbert
sequencing or chemical
sequencing method
Sanger’s Method/ Sanger’s Sequencing/ Chain
Termination Method

• History
• It was developed by Frederick Sanger along with Andrew Coulson
in 1977.
• They were awarded Nobel prize in 1980 on this achievement.
• Frederick Sanger double Nobel prize winner.
• First Nobel Prize -1958: Sequence of Amino Acid in insulin.
• Second Nobel Prize -1980: Father of Genomics.
Requirements

• Single strand of DNA to be sequenced.


• Taq DNA polymerase.
• Primer.
• DNTPs four different types (dATPs, dGTPs, dCTPs, dTTPs)
• Dideoxynucleoside triphosphats four different types (ddATPs,
ddGTPs, ddCTPs, ddTTPs)
Procedure

• Same as DNA Replication process.


• Before the DNA sequencing. It has to be denatured into single
strands. Using heat as only one strand act as template.
• Now the template strands tagged with a known sequence at 3’ end so
that a complimentary Primer can bind on the known sequence.
• Tagged target DNA, Primer free normal dNTP’s and Tag Polymerase
dissolved in an appropriate concentration and equally added in 4
tubes.
• But each tube containing different ddNTP each at about one
hundredth the concentration of the normal nucleotide.
As the DNA is synthesized,
nucleotides are added on to the
growing chain by the DNA
Polymerase.
However when ddNTP is
incorporated into the chain in
place of a normal nucleotide
which result in a chain
termination event.
Once these reactions are
completed the DNA is once
again denatured in preparation
for gel electrophoresis.
• The contents in each of 4 type of tubes are run in separate lanes on
polyaccrylamide gel in order to separate the different sized bands from one
another.
• Then the gel is exposed to UV light/x rays.
• The sequence read from the gel is complimentary to the actual template
DNA.
• Uses of Sanger’s Sequence
• Region up to about 900 base pairs in length sequenced using this method
• In Human Genome project sanger sequencing was used to determine the
sequence of relatively small fragments of human DNA
• Sanger sequencing is still in wide use for the sequencing of individual pieces
of DNA, such as fragments used in DNA cloning
MAXAM GILBERT DNA SEQUENCING

• In 1976_1977 , Allan MAXAM and Walter GILBERT developed a DNA


sequencing method .
• It is a method by which the sequence of DNA fragment is identifed
by using chemicals , that’s cut DNA at specific points.
• Also called chemical cleavage method of DN
PROCEDURE

• STEP 1
• END LABELLING:
• The procedure involves the radioactive labeling of the 5′-P ends of
double-stranded DNA (dsDNA) with 32P-dATP
using polynucleotide kinase. Then, the DNA is denatured
with DiMethyl SulfOxide (DMSO) at 90°C and the resulting ssDNA
molecules are segregate
S
An equal volume of 4 different ssDNA
samples is taken into 4 different tubes
each containing 4 different chemicals
• If we see the sequence on gel from 5 prime to 3 prime,
and compare it, it is the same sequence that we have at
first :Fragment sequence that was selected :Fragment
sequence identified from the Gel
ADVANTAGES. DISADVANTAGES:

• Directly read purified DNA. • Use of toxic chemicals


• used to analyze DNA-Protein • Setup is quit complex
interaction

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