|



 


 

| 

The term DNA sequencing refers to
sequencing methods for determining
the order
f the nucleotide bases- adenine,
guanine, cytosine, and thymine- in a
molecule of DNA
  |
 | 





K axam and Gilbert method

K
anger dideoxy method
axam and Gilbert sequencing
method

anger dideoxy sequencing
method
 
  | 
|
 




K 
NCING
K NAN
NCING
K  D WAVGID
K 
NCLA

NCING
ã  

 ã
ã 

A sequencing primer is hybridized to a single-stranded C
amplicon that serves as a template, and incubated with the
enzymes, DNA polymerase, AT sulfurylase, luciferase, and
apyrase as well as the substrates, adenosine 5' phosphosulfate
(A
, and luciferin.
  
The first deoxribonucleotide triphosphate (dNT is added to the
reaction. DNA polymerase catalyzes the incorporation of the
deoxyribo-nucleotide triphosphate into the DNA strand, if it is
complementary to the base in the template strand. ach
incorporation event is accompanied by release of pyrophosphate
(i in a quantity equimolar to the amount of incorporated
nucleotide.
 !
AT sulfurylase converts i to AT in the presence of adenosine 5'
phosphosulfate (A
. This AT drives the luciferase-mediated
conversion of luciferin to oxyluciferin that generates visible light in
amounts that are proportional to the amount of AT. The light
produced in the luciferase-catalyzed reaction is detected by a charge
coupled device (CCD chip and seen as a peak in the raw data output
(yrogram. The height of each peak (light signal is proportional to
the number of nucleotides incorporated.
 "
Apyrase, a nucleotide-degrading enzyme, continuously degrades
unincorporated nucleotides and AT. When degradation is complete,
another nucleotide is added
 #
Addition of dNTs is performed sequentially. It should be noted that
deoxyadenosine alfa-thio triphosphate (dAT
 is used as a substitute
for the natural deoxyadenosine triphosphate (dAT since it is efficiently
used by the DNA polymerase, but not recognized by the luciferase. As
the process continues, the complementary DNA strand is built up and
the nucleotide sequence is determined from the signal peaks in the
yrogram trace
 | ã  

Nanopore:- A nanopore is simply a small hole,

of the order of 1 nanometer in internal
diameter.

 $

 | ã 
 

 ã
ã 

T 1
Design and synthesis of modified
nucleotides

O
  


n 7-position of A (6-amino-
hexylamino
5-postion of T (Biotin
T*>A*>G>C
V
  

 

To enhance discrimination between two purines
and two pyrimidines because these generate
similar electronic signature

T 2
DNA extension reaction using modified
nucleotides.
odified dAT and dTT
and unmodified dCT and dGT

T 3
emoving from resulting ds DNA, the ss DNA
containing modified nucleotides

T 4
ss DNA pass through a pore of suitable
diameter (nanopore

T 5
ach type of nucleotide in the DNA has unique
electronic signature and this is used to
determine the sequence of nucleotides in ss
DNA
 (  

|%



 &' 
|

 
W is a cylindrical hole just a few tens of
nanometer in diameter, perforating a thin film
supported by a transparent substrate
 
 LINKD NCLTID

tep 1: Fluorescent phospholinked labeled nucleotides

are introduced into the W.

tep 2: The base being incorporated is held in the
detection volume for tens of milliseconds, producing a
bright flash of light.

tep 3: The phosphate chain is cleaved, releasing the
attached dye molecule.

tep 4-5: The process repeats.
  | 

This method of sequencing exploits 3¶65¶

activity of exonuclease
 ã
ã 

T 1
labeling the nucleotides with base specific
tags suitable for fluorescence detection

T 2

electing a desired single stranded fragment of
DNA and synthesizing its second strand with
the help of DNA polymerase

T 3

uspending the single DNA fragment in a
flowing sample stream

T
4 and 5

equentially cleaving labeled bases from the
free end of the DNA fragment using an
exonuclease, and Detecting and identifying the
cleaved, labeled bases as they flow through a
focused laser beam
 |ãã| ( 

  | 
|
 



K Genomic medicine
K etagenomics
K Comparative genomics
 (  
 |

 ' ))*
|
+   
  
|  , -%!#- ./ *0/ !*


 ' ))#|

 

 , -#*!-!0")

!
1-' ))* ,  &


|
!

- '"-"#2 ".

" +3344443 33456
7 ))*"/#.

# +3348 ,3483ã,

/ +33444,
&3
ã96 7*"#"
* +33444
,&3
,&  
.:
:- |1,-:,-$,,- 1;- 
<<- 1;,-$ ,- 4,'2.2

 
|
+,  
<
,
<&


  





2<  -  =- ã ,='22.
|
 
+|
  $   &
ã,  
- .-!/! !/#
) 

<, - 
&-
, 
,-| &ã,>
$-: 
9-ã,  -,8$,   
&, ?' ))#|< << ,, 

! 
!!'2+*@
+))2!3,3*)'  

 
' ))*|
 

   ,
4 ,&&, :
,<9,&
$- )2-#. # #

 444<&33A A8,


, <

!444 
33
A;,A& A4

 A
A ))! <