DNA

TECHNOLOGY
SUCHANUN SRIKAEW
GENETIC
ENGINEERING
1) CUTTING & PASTING DNA
• Use restriction enzymes cut out the gene
of interest
RESTRICTION ENZYMES
RESTRICTION ENZYMES

• blunt ends

• sticky ends
RESTRICTION ENZYMES
1) CUTTING & PASTING DNA
• Separately digest (cut) the gene fragment
and the plasmid with EcoRI
1) CUTTING & PASTING DNA
• Separately digest (cut) the gene fragment
and the plasmid with EcoRI
1) CUTTING & PASTING DNA

• Then, combine the

fragments with DNA ligase
RECOMBINANT DNA
• The technology used for making artificial
DNA by combining different DNA from
different sources
PLASMID

• Origin of replication
• Multiple cloning site
• Selectable marker gene
2) BACTERIAL TRANSFORMATION
• Plasmids and other DNA can be introduced
into bacteria
3) BACTERIAL SELECTION
• Use antibiotic selection and DNA analysis
methods to identify bacteria that contain
the plasmid
POLYMERASE
CHAIN
REACTION

• The technique to make

many copies of a specific
DNA region in vitro
 1) DENATURATION
• (96°C): Heat the reaction strongly to separate,
or denature, the DNA strands
 2) ANNEALING
• (55 - 65°C): Cool the reaction so the primers
can bind to their complementary sequences
3) EXTENSION

• (72°C): Raise the

reaction temperatures
so Taq polymerase
extends the primers,
synthesizing new
strands of DNA
POLYMERASE CHAIN REACTION
• The cycle repeats 25-35 times which
generally takes 2-4 hours, depending on
the length of the DNA region being copied
GEL
ELECTROPHORESIS
GEL ELECTROPHORESIS

• The technique used

to separate DNA
fragments according
to their size
AGAROSE GEL
• Gels for DNA separation are often made
out of a polysaccharide
GEL ELECTROPHORESIS
• DNA samples are loaded into wells at one
end of a gel, and an electric current is
applied to pull them through the gel
 GEL ELECTROPHORESIS
• When a gel is stained with a DNA-binding dye,
the DNA fragments can be seen as bands

• Ethidium
bromide
RFLP ANALYSIS
HUMAN GENOME
DNA SEQUENCING
• The process of determining the sequence
of nucleotide bases (As, Ts, Cs, and Gs) in
a piece of DNA
SANGER SEQUENCING

• The target DNA

is copied many
times, making
fragments of
different lengths
 SANGER SEQUENCING
• Fluorescent nucleotides mark the ends of
the fragments and allow the sequence to
be determined
SANGER SEQUENCING
NEXT-GENERATION SEQUENCING
• new, large-scale approaches that increase
the speed and reduce the cost of DNA
sequencing