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Biotechnology -- Chap. 16.
The use of biological systems for the
production of materials (most work is in the
field of Genetic Engineering)
Genetic engineering
• process of altering biological systems by the
purposeful manipulation of DNA
– introduce specific foreign pieces of DNA into
host cells (often bacteria or viruses) which is
then replicated by the host cells
– as the cell divides, a clone (exact copy) is
produced
– cells containing the new DNA can be grown
in any quantity and therefore, so is the
protein transcribed by that DNA.
The Process
• The hard part is to “convince” the host cell to
accept the foreign DNA
– this is done by attaching the foreign DNA to a
carrier DNA molecule called a vector.
– vectors are often bacteria’s plasmid (once
together they are now recombinant DNA)
• Plasmids are molecules of DNA that are found in
bacteria separate from the bacterial chromosome.
They:
• are small, carrying one or a few genes
• are circular
• self-replicating
• This tiny but mighty plasmid molecule is the
basis of recombinant DNA technology.
Restriction enzymes (also called restriction
endonucleases)
• enzymes that cut the phosphatebackbones of DNA at specific base
sequences (normally 4-6 bases) called
restriction sites.
• exist naturally in bacteria in order to cut
apart invading viral DNA
• cut parts of DNA are called restriction
fragments
• naming: ex) EcoRI
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–
–
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1st letter is for genus - (Escherichia)
2nd letter/s is for species - (coli)
3rd letter is for the strain of the organism (R)
Roman numerals at the end typically
indicates the order of discovery (I)
Recombinant DNA video
Splicing and Cloning DNA Molecules (Fig.
16.2 in text)
• a restriction fragment of DNA can be
duplicated by inserting it in a vector DNA
molecule
• most common vectors are plasmids in bacteria
and DNA viruses
• first, the vector and foreign DNA are cleaved by
the same restriction enzyme in order for the
sticky ends of both to match and then mixed
together
• The enzyme ligase is added to join the vector
and foreign DNA together making the foreign
DNA an integral part of the plasmid
• This new plasmid is then taken up by other
bacteria cells by a process called transformation
• Identification: Often the foreign DNA that was
just inserted has other segments of DNA that
identify it, such as resistance to antibiotics and
the ability to produce color.
Steps in Cloning a Gene
DNA Sequencing
• the determination of the precise sequence
of nucleotides in a sample of DNA.
• dideoxynucleotides are synthetic
nucleotides (4 types, ddATP, ddGTP,
ddCTP, ddTTP) that lack the -OH at the
3′ carbon atom and are each labeled with
a "tag" that fluoresces a different color
• chain elongation proceeds normally until
by chance, DNA polymerase inserts a
dideoxynucleotide instead of the normal
deoxynucleotide
• Each of the four dideoxynucleotides
fluoresces a different color when
illuminated by a laser beam and an
automatic scanner provides a printout of
the sequence