Genome Sequencing

Timeline of large-scale genomic DNA Sequencing

• Isolation of
DNA/specific portion
• Shear into
pieces/fragments
• Amplify
• labeling
• Sequencing fragments
• Generate Reads
• Assemble reads in
order
 Isolation of DNA
• Lyse – Breaking the cell
membrane
• Bind – Binding of nucleic
acid to silica gel membrane
• Wash - washing the nucleic
acid bound to the silica gel
membrane to remove
impurities
• Elute – Removing the DNA
from silica gel membrane
https://www.youtube.com/watch?v=qfa0hi6s35E
 Fragmentation
• Longer sequences subdivided into smaller
fragments.

• Sequencing can only be performed for fairly short

strands (100 to 5000 base pairs)
• Quality of the base identification decreases with
length.
• Three methods – Physical, Chemical and enzyme
assisted.
 Physical method
• Physical methods like ultrasonication, acoustic
shearing use different frequency of sound
wave to shear DNA.
• The fragments obtained are nonspecific.
• Hydrogen bond in double helix as well as the
oxygen carbon bond broken.
 Enzymatic Fragmentation
• Enzyme assisted fragmentation uses Endonuclease
enzymes.
• Involved in defense mechanism.
• Extracted from several bacteria.
• These enzymes are site specific, the sequence of end part
of fragments are known.
• Can break both strands uniformly or leave sticky ends
• DNA fragment produced are called restriction fragment
• Human genome produces millions of fragments
• Fragments are separated by electrophoresis
Enzymes and fragmentation
 Amplification
• Process to increase the number of fragments
• Helps in isolation & identification of fragments
by gel electrophoresis
• Polymerase chain reaction(PCR)
• Cloning(DNA recombinant Technology)
 PCR
• The method for selective amplification is called
the polymerase chain reaction (PCR).
• Kary B. Mullis received Nobel Prize in 1993.
• PCR amplification requires DNA polymerase, a
pair of short, synthetic primers & nucleotides
• DNA polymerase obtained from heat resistant
bacteria
• Primers complementary in sequence for each
strand of the fragment.
• The three steps are
Denaturation, annealing and
elongation
• Denaturation is unzipping of
DNA. Temperature 95oC
• Annealing of primer at
temperature 50-60oC
• Elongation of chain in 5' direction
by addition of nucleotides
• First cycle there is a pair of
parent strands and a pair of
synthetic strand
• At the end of 25th cycle 3.4x107
fragments.
https://www.youtube.com/watch?v=ThG_02miq-4
 DNA Cloning
• Plasmids are special DNA
in certain bacteria
• Selected fragment of
DNA can be inserted into
the fragment by cut and
paste mechanism
• Uses restriction enzymes.
• The recombinant DNA
amplifies when insrted in
bacteria.
 Amplification and isolation

•The fragments of DNA are isolated by lysis with

restriction enzymes.
•The fragments obtained are used for further analysis.
Additional information……
 Sequencing
• Identification of base sequences in the
fragments
• Methods like Sanger Sequencing, Pyro
sequencing, Illumina sequencing etc employed
• Sanger method sequenced the first genome