Sub-discipline of genetics dedicated to

Genomics the study of structure and function of

genome of an organism)

Structural Functional Comparative

Genomics Genomics Genomics

Structural genomics refers to the
initial phase of genome analysis
✓ Genome Sequencing
✓ Construction of genetic and
physical maps of a genome
✓ Identification of genes
✓ Annotation of gene features
Refers to the analysis of Used to compare genomes of
global gene expression different organisms.
and gene functions in a ✓ Includes comparison of gene
genome. number, gene location, and
gene content
✓ Transcriptome analysis ✓ Gene order
✓ How sets of genes work
together to form
metabolic, regulatory,
and signaling pathways
within the cell.
 DNA Sequencing

• DNA sequencing is the process by which the precise

order of nucleotides in a piece of DNA can be
determined.

• All the information required for growth and

development of an organism is encoded in the DNA
of its genome.

• So, DNA sequencing is fundamental to genome

analysis and understanding the biological process in
general.
Historical Timeline
 Sequencing Methods

1. Chain Termination Method: Fredrick Sanger

2. Chemical Sequencing Method: Maxam and Gilbert

First Generation Sequencing Methods

 Maxam Gilbert Method: Chain degradation Method

• It is a method by which the sequence of a DNA fragment is

identified by using chemicals that cut DNA at specific points.

• Developed by Allan Maxam and Walter Gilbert in 1976-77

DMSO
 2 Separation of single stranded DNA

The two strands are separated by gel electrophoresis.

One of the strands contains more purines than the others

and is therefore slightly heavier and runs more slowly
during electrophoresis.
 3 Radioactive labelling

• Take one fragment band from the gel.

• Remove the Phosphate from the 5’ end:

• Incorporate Radioactive Phosphate P32 enzymatically.

 4 Chemical reaction and Cleavage

A+G A T+C C
5 Electrophoresis and Autoradiography
 Sanger’s Method: Chain termination Method/Dideoxy method

• Most widely used method: basis of automated sequencing

• Developed by Frederick Sanger and colleagues in 1977.

• Nobel Prize in 1980

• It is a PCR based method.

• A modified DNA replication reaction.

• Growing chains terminated by dideoxynucleotides.

M13 bacteriophage
or plasmid vector
DNA Pol I

5’- 3’ polymerase
5’-3’ exonuclease
3’-5’ exonuclease

Klenow Fragment

Seqeuenase
Primer needed
Advantages n disadvantages
 Cycle Sequencing

• Cycle sequencing is a method used to increase the sensitivity of the DNA

sequencing process and permits the use of very small amounts of DNA starting
material.

• This is accomplished by using a temperature cycling process similar to that

employed in the polymerase chain reaction.

• In cycle sequencing, a reaction is taken through several steps designed to

prepare the template for copying, allow for initiation of DNA synthesis, and
generate the terminated DNA chains needed for electrophoresis and sequence
determination.

• The first step in the process is called denaturation, in which double-stranded

template DNA is converted into its single-stranded form. This is accomplished
by heating the template to between 94 °C and 98 °C, a temperature high enough
to break the hydrogen bonds between the complementary bases holding the two
strands together.
• As a single-stranded molecule, the template's bases are now exposed, and are free to
interact with the sequencing primer. The primer, in a step called annealing, locates and
attaches itself to its complementary site on the template. Thus, Ts bind to As and Cs bind
to Gs. However, primer annealing will only occur at a temperature where hydrogen
bonds can form between the primer and template strands, usually between 40 °C and
65 °C.

• In the final step of the reaction, DNA polymerase extends the annealed primer by
sequentially adding on to its end bases that are complementary to those on the template.
It is during this extension step of a DNA sequencing reaction that random incorporation
of a dideoxynucleotide can occur, terminating chain growth.

• All three of these steps, taken together, represent one round, or cycle, of a DNA synthesis
reaction. By repeating a cycle over and over again, the amount of each fragment made in
the reaction can be substantially increased.

• Since each fragment carries fluorescent dyes, increasing the number of copied fragments
also increases the strength of the fluorescent signal. Cycle sequencing, therefore, greatly
improves the sensitivity of the sequencing reaction, and even very small amounts of
starting DNA sample can be used as template.
Automatic Sequencing