LIST OF CONTENTS


 HISTORY OF DNA SEQUENCING
 INTRODUCTION
 WHAT EXACTLY IS DNA
SEQUENCING….??
 DNA STRUCTURE
 IN THE BEGINNING
 HOW IS DNA SEQUENCING
PERFORMED..??
 DIFFERENT METHODS FOR DNA
SEQUENCING
 SANGER SEQUENCING
 MAX-GILBERT SEQUENCING
 APPLICATION
 ADVANTAGES
 DISADVANTAGES
 CONCLUSION
HISTORY OF DNA SEQUENCING

 The sequencing of DNA
molecules began in the 1970s with
development of the Maxam-
Gilbert ethod, and later the
Sanger method.
 Originally developed by
Frederick Sanger in 1975, most
DNA sequencing that occurs in
medical and research laboratories
today is performed using
sequencers employing variations
of the Sanger method.
HISTORY

 INTRODUCTION

 The term DNA sequencing refers to sequencing methods
o
fr determining the order of the nucleotide bases - adenine,
guanine, cytosine, and thymine - in a molecule of DNA.
Knowledge of DNA sequences has become indispensable for
basic biological research, other research branches utilizing
DNA sequencing, and in numerous applied fields such as:
 Diagnostic,
 Biotechnology,
 Forensic Biology And
 Biological Systematics.
 WHAT EXACTLY IS DNA
SEQUENCING….??

 The very basic unit of the human genome is a single
DNA nucleotide. This nucleotide is extremely small and
is made up of minuscule atoms, which creates a challenge
for even an advanced microscope to be used for
detection.
Researchers still, however, need to be able to determine the
sequence of bases in DNA that make up the human
genome. As such, DNA sequencing has been
developed but the process itself is a seemingly
complex one.
 DNA sequencing involves the determination of the
order of DNA bases.
 DNA STRUCTURE

 In a strand of DNA, there are some simple units known as
nucleotides. These nucleotides have a 'backbone' that consists of
sugars and a phosphate group. The DNA bases can be one of
four kinds and they are attached to these sugars. These bases
hold the important and unique genetic information for body.
These bases are:
 Adenine (A)
 Thymine (T)
 Cytosine (C)
 Guanine (G)
 IN THE BEGINNING


 The very first methods used for DNA sequencing were
created
in the 1970s.
 During this decade, researchers were only able to sequence
a small number of base pairs, .
 By 1990, things had improved somewhat but the
number of laboratories able to sequence a hundred
thousand bases was still few.
 Not only that, but the cost of sequencing itself was extremely
high and impractical.
 Fortunately, there have been vast improvements since
then, particularly in terms of technological advancement.
 
 Better still, automation has made the process much faster
and a
great deal more practical.
 Now, individual genes are sequenced on a regular basis
n
ad can be done quickly and affordably for laboratories.
 In fact, some laboratories are sequencing more than a
hundred million bases in a given year
 HOW IS DNA SEQUENCING
PERFORMED..??

 DNA sequencing involves the process of figuring out h te
precise order of the four bases found in one piece of
DNA.
 The DNA is really just a template that is used to
create a series
of fragments.
 The fragments differ in length by one base and they are
separated by size before the bases are identified, which then
effectively recreates the original DNA sequence.
 Each person has twenty-three pairs of chromosomes -
one copy
of the human genome.

Because technology has limitations, we are limited in
ho
wmany bases can be read at one time.
 Therefore, we can't just read each base from one
end of a chromosome to the other. To make it feasible,
the chromosome is cut down into smaller fragments.
 SOME EXAMPLES….
Exp
Exp
.1
2


 Exp


2
.3

1
3
 DIFFERENT METHODS FOR DNA
SEQUENCING

 Maxam-Gilbert sequencing



Chain-termination methods
Next-generation methods:
 Massively parallel signature sequencing (MPSS)

 Basic
Polony Methods:
sequencing
 454 pyrosequencing
 Illumina (Solexa) sequencing
 SOLiD sequencing
 Ion Torrent semiconductor sequencing
 DNA nanoball sequencing
 Single molecule real time (SMRT) sequencing
 
SANGER SEQUENCING
OR
CHAIN TERMINATION
METHOD
 HISTORY

 Developed by Frederick Sanger and colleagues
in 1977,
 it was the most widely used sequencing method
for approximately 25 years after its discovery.
 He got NOBEL PRIZE in 1980.
 INTRODUCTION

 It is method to find out the nucleotides Sequence of
unknownDNA strand.
 More recently, Sanger sequencing has been upgraded as "Next-
Generation“ sequencing methods, especially for large scale
genome analyses and for obtaining especially long DNA
sequence reads (>500 nucleotides).
 BASIC PRINCIPLE



This method generally is an In-Vitro synthesis of DNA strand
and by using terminators (di-deoxynucleotide) the growing
strand terminates at specific site.
 Upon termination the strands are overlap to got original
sequence of unknown DNA Strand.

3’ T TT T 5’

ddATP in the
ddA reaction:
anywhere there’s
a T in the template
ddA
strand,
occasionally a ddA
ddA will be added to
the growing strand
ddA
 REQUIREMENTS

 Single Stranded template
 Primer
 DNA polymerase
 Di-Deoxynucleotide
 The 3′-OH group necessary for
formation of the phosphodiester bond
is missing in ddNTPs)
 Every nucleotide have its specific
ddNTP form i.e., ddATP, ddGTP etc
 PROCEDURE

 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Gel electrophoresis
 Cont….

 The DNA template is treated with heat so that it becomes
single
stranded
 A short, single-stranded primer which is radioactively labeled
is added to the end of the DNA template
 Add template DNA and primer in 4 Tubes.
 Now add ddNTPs In tubes in the way that single tube
contain
one type of ddNTP.
 Extension is start and band formed of various sizes.
 The fragments of DNA are separated by electrophoresis
 Overlap these sequences to find out sequence of Target
DNA.
