Previous concepts

• Building block of DNA are

nucleotides
• Double helix
• Both strands are anti parallel
• Million of base pair lie one
over other
• DNA read from 5’ to 3’
• Length measured in base
pair units
• Kb = 1000 base pair
 Organization of DNA
• DNA strand is longer than
the nucleus
• Smallest DNA is
14000 μm.
• Average size of nucleus
6 μm
• DNA is packed as
Chromosome
• Packing ratio – Length
of DNA/length of
Chromosome
 Replication
• Biological process of
producing two identical
replicas of DNA from one
original molecule.
• DNA make copy of itself
• Each strand acts as
template
• can also be performed in
vitro
• Double stranded molecule gets converted into
two identical double stranded molecule/DNA
 Essentials
• A parent strand as template
• Nucleotides containing bases adenine,
guanine, cytosine & thymine
• RNA Primer – oligonucleotide containing upto
30 bp
• DNA polymerase
• Some proteins and enzymes like helicase,
ligase
Primer
Replication begins at 3’end of each strand
Bases are added one at a time
Process continues till the strand is completed
 Questions
• The process of replication begins from ----------
end of DNA
• The enzyme which helps in extension of strand
is -----------
• Primer is essential to ------------------
• Nucleotides are of ----------- types
 DNA Sequencing
• Process to find base pair sequence in a given
DNA
• DNAs present in each cell in an organism are
same
• Genome sequence is the sequence of all DNA
present in an organism’s cell
• Generally the germ cell (egg or sperm) is
considered because it is haploid
 Why sequencing
• DNA sequences required for
• Basic biological research
• Research in evolutionary pattern
• Applied fields such as medical diagonsis,
Biotechnology, Forensics, virology
 Methods
• Classical Methods – Maxam Gilbert method,
Sengar method
• Accurate
• Slow, read short fragments, expensive, harmful
chemicals used.
• De Novo or Next Gen sequencing – Shotgun
method, illumina sequencing methods
• Faster, longer fragments can be read, lesser
accuracy, cheaper
• Isolation of
DNA/specific portion
• Shear into
pieces/fragments
• Amplify
• labeling
• Sequencing fragments
• Generate Reads
• Assemble reads in
order
 Challenges
• Read size
• Time
• Accuracy
• Cost