Download DNA Cloning - MrMsciences

DNA Cloning
Cloning
• a line of genetically
identical cells or
individuals derived
from a single ancestor
• produces many
copies of a piece of
DNA
• uses a little fraction
as gene of interest
• cultivates a large
amount for studying
functions
Functions
• Biological
• determine the DNA
sequence
• ultimate information
• regulation
• Pharmaceutical
• manufacture
medicines
• Clinical
How it Works
• Combine gene of
interest and bacterial
plasmid
• Recombinant DNA
• DNA from two or
more different
sources that have
been joined together
to form a single
molecule
• Amplification and
identification
Zoom In!!!
• Insertion, the most
critical step
• made possible by
restriction
endonucleases(aka.
restriction enzymes)
• recognize specific
DNA sequences
called restriction sites
• cut the DNA at
specific locations
• Restriction fragments
Restriction Enzyme
• defend against viral attack by
breaking down the DNA
molecules of infecting viruses
• cleave the sugar-phosphate
backbones of DNA to
produce sticky ends
• short single-stranded regions
• form hydrogen bonds with
complementary sticky ends
on any other DNA molecules
cut with the same enzyme
• kind of like glue when you fix
something
Cloning Vectors
• DNA molecules into
which a DNA
fragment can be
inserted to form a
recombinant DNA
molecule for the
purpose of cloning
• plasmid cloning
vectors
• contains 2 unique
genes: ampR gene
and lacZ+ gene
The Two Genes
• ampR gene
• encodes an enzyme
that breaks down the
antibiotic ampicillin
• lacZ+ gene
• encodes ßgalactosidase
(hydrolyzes the sugar
lactorse and synthetic
substrates)