For medical microbiology students under TU Nepal

1. Neeraj Sharma
MSc. Medical Microbiology
2nd year

2.  The process of determining the order of the
nucleotide bases along a DNA strand.
 In 1977 two separate methods of sequencing of
DNA were developed.
 Chain termination method and Chemical
Degredation method.
 Chain termination method is commonly used
today

3.  Also called dideoxynucleotide or Sangers
method.
 This method is based on the principle that
single stranded DNA molecules that differ in
length by just a nucleotide can be separated
from one another using polyacrylamide gel
electrophoresis.

4.  A DNA template.
 A primer tagged with a mildly radioactive
molecule or light emitting chemical.
 DNA Polymerase
 Deoxynucleotide (G, A, C, T)
 Dideoxynucleotide (ddG, ddA, ddC, ddT)

5.  Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Gel electrophoresis

6.  Denaturation of the
Double stranded
DNA into a single
stranded DNA
template.

7.  Carried out in four set
of test tubes.
 Each tube contains
DNA fragments, DNA
primer, nucleotides,
DNA polymerase.
 1% of the total
solution ddA, ddG,
ddC and ddT in
separate tubes.

10.  The DNA fragments
are separated by size
with polyacrylamide
gel electrophoresis ,
and because a radio
active DNA primer
was used , the
position of the band in
the gel is determined
by autoradiography.

11.  The autoradiogram is
then read starting at
the bottom where the
DNA fragments are
the shortest.
 These bands at the
bottom represent the
sequence of DNA
bases nearest to the
primer.

12.  The sequence is read
one base at a time in
the 5’ to 3’ direction.
 The sequence being
read is
complementary to the
DNA fragment. The
sequence of short
fragment is
 5’ A G C C T A G A C
T 3’

13.  Other methods.
Shot gun sequencing.
Pyrosequencing.

14.  Determine the sequence of individual gene,
larger genetic regions, full chromosome or
entire genome.
 Provide the order of individual nucleotides in
DNA RNA isolated from cells of animals plants
bacteria etc.

15.  Molecular biology - studying the genome
itself, how proteins are made, what proteins
are made, identifying new genes and
associations with diseases and phenotypes, and
identifying potential drug targets
 Evolutionary biology - studying how different
organisms are related and how they evolved
 Helpful in ecology, epidemiology,research and
other fields.