Course Material Unit: Genomics

PhD Biotechnology/Biochemistry

BASICS OF GENOME
SEQUENCING

- Dr Kumar Sachin
Faculty-Biosciences
 INTRODUCTION

• Rapid and efficient methods for DNA

sequencing were first devised in the mid-1970s.
• There are two different procedures of
sequencing DNA chains that were developed
almost contemporary:
– The chain termination method (Sanger et al., 1977)
– The chemical degradation method (Maxam and
Gilbert, 1977)
 The Chain Termination Method
• The method is based on the principle that single-stranded
DNA molecules that differ in length by just a single
nucleotide can be separated from one another
by polyacrylamide gel electrophoresis. This means that it
is possible to resolve a family of molecules, representing
all lengths from 10 to 1500 nucleotides, into a series of
bands.
• The sequence of a single-stranded DNA molecule is
determined by enzymatic synthesis of complementary
polynucleotide chains, these chains terminating at specific
nucleotide positions
 Sanger’s Method
• The first step: preparation of identical single-stranded DNA molecules.
A short oligonucleotide is annealed to the same position on each
molecule, that act as the primer for synthesis of a new DNA strand
complementary to the template.

• The strand synthesis reaction, which is catalyzed by a DNA

polymerase enzyme requires the four deoxyribonucleotide
triphosphates |(dNTPs) as substrates; besides, a small amount of
a dideoxynucleotide (e.g. ddATP) is also added to the reaction mix. The
polymerase enzyme does not discriminate between dNTPs and ddNTPs,
so the dideoxynucleotide can be incorporated into the growing chain,
but it then blocks further elongation because it lacks the 3′-hydroxyl
group needed to form a connection with the next nucleotide.  

Resource: https://www.youtube.com/watch?v=UPgoZcN8_zU
 Maxam-Gilbert Method

• The chemical degradation method:

The sequence of a double-
stranded DNA molecule is determined by
treatment with chemicals that cut the molecule
at specific nucleotide positions.
 High-throughput sequencing

• Conventional DNA sequencing suffers from the

limitation that only a few hundred bp of
sequence can be determined in a single
experiment.
• Therefore, there have been attempts to make
sequence acquisition more rapid by devising
new sequencing methodologies
 Pyrosequencing

The strand synthesis reaction is carried out in the absence of dideoxynucleotides.

Each dNTP is added individually, along with a nucleotidase enzyme that degrades the
dNTP if it is not incorporated into the strand being synthesized. Incorporation of a
nucleotide is detected by a flash of chemiluminescence induced by the pyrophosphate
released from the dNTP. The order in which nucleotides are added to the growing strand
can therefore be followed.