Biotechnology
3A1: DNA, and is some cases RNA, is the primary source of heritable
information.
Pair Share
•What is biotechnology?
•How long has biotechnology been taking
place?
•Is biotechnology and genetic engineering
the same thing?
•What does it mean to engineer something?
Biotechnology vs Genetic engineering
• Biotechnology: manipulation of organisms or their components to make
useful products
• Long history: selective breeding of farm animals; using microorganisms to make wine
and cheese
• Genetic Engineering: direct manipulation of genes for practical purposes
• Launched a revolution in biotechnology  expanding the scope of potential
applications (agriculture, criminal law, medical research)
• Examples:
•
•
•
•
Recombinant DNA: DNA from two sources combined together
Restriction Enzymes: essentially specific DNA cutters
Gel electrophoresis: technique used to separate DNA by size
Polymerase chain reaciton (PRC): technique used to quickly make copies of DNA
Review – What is a gene????
• Chromosome = DNA
• DNA = multiple genes
• Gene = segments of DNA that codes for a protein
• Protein = 3D structure of amino acids folded into a specific
way that provides specific structure and function
• To work directly with specific genes, scientists have
developed techniques to clone DNA
Gene Cloning
• Gene Cloning: the produciton of multiple copies of a single gene;
two basic purposes: to make copies of a particular gene and to
produce a protein product
• One common approach to gene cloning is to use bacteria
• Review of bacterial DNA
• large circular molecule of DNA
• Plasmid: small circular DNA molecule that replicates separately from the bacterial
chromosome – contains a small number of genes beneficial to the bacteria in
particular environments, but not needed for survival and reproduction
General Steps of Gene Cloning
• Isolate plasmid and gene of
interest
• Insert gene of interest into plasmid
• Return plasmid back into bacteria
• Allow bacteria to reproduce 
reproduced bacteria will also
replicate gene of interest
• Products can be used to do further
research of the particular gene,
insert the gene into other
organisms (recombinant DNA), use
to clean up toxic waste, used for
medical purposes
Bacterium
1 Gene inserted into
plasmid
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
DNA of
chromosome
2 Plasmid put into
bacterial cell
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
Interest
Protein expressed
by gene of interest
Copies of gene
Basic
Protein harvested
4 Basic research and
various applications
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Restriction site
Using Restriction Enzymes to Make Recombinant DNA
• Restriction enzymes: Enzymes that cuts DNA at specific locations;
also called restriction endonucleases
• Recognizes specific DNA sequence called a restriction site
• Cuts DNA strands at precise points within the restriction site
• Most restriction sites are symmetrical, sequences of nucleotides is
the same on both strands when read in the 5’3’ direction
• Results in restriction fragments – pieces of DNA cut by restriction
enzymes
• All copies of a particular DNA molecule always yield the same set of
restriction fragments when exposed to the same restriction enzyme
• Most useful restriction enzymes cut in a staggered manner resulting
in sticky ends
• Sticky ends: short extensions of DNA that can form hydrogenbonded base pairs with complementary sticky ends on any other
DNA molecule cut with the same restriction enzyme
• DNA cut but the same restriction enzymes can be recombined to
form recombinant DNA
• DNA ligase: enzyme that catalyzes the formation of covalent bonds
that close up the sugar-phosphate backbones of DNA; think back to
Okazaki gragments during replication
• https://www.youtube.com/watch?v=pDHCHa1C85Y
DNA
1
5
3
3
5
Restriction enzyme
cuts sugar-phosphate
backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Cloning a Eukaryotic Gene in a Bacterial
Plasmid (More Details)
• Cloning Vector: DNA molecule that can carry foreign DNA into a host
cell and replicate there
• Bacteria plasmids are widely used as cloning vectors because they are
easily isolated from bacteria, manipulated to form recombinant
plasmids by inserting foreign DNA, and reintroduced into bacterial
cells; they also multiple rapidly
• Due to the difference in the way genes are expressed in eukaryotic
cells vs prokaryotic cells, it is difficult to get a eukaryotic gene to be
expressed in bacteria.
• Alternative is to use yeast, which is a eukaryotic cell, but also contains
a plasmid
Cloning a Eukaryotic Gene in a Bacterial
Plasmid
• In gene cloning, the original plasmid is called a
cloning vector
• A cloning vector is a DNA molecule that can carry
foreign DNA into a host cell and replicate there
Producing Clones of Cells Carrying
Recombinant Plasmids
• Several steps are required to clone the hummingbird
β-globin gene in a bacterial plasmid:
• The hummingbird genomic DNA and a bacterial plasmid are
isolated
• Both are digested with the same restriction enzyme
• The fragments are mixed, and DNA ligase is added to bond
the fragment sticky ends
Animation: Cloning a Gene
• Some recombinant plasmids now contain hummingbird DNA
• The DNA mixture is added to bacteria that have been
genetically engineered to accept it
• The bacteria are plated on a type of agar that selects for the
bacteria with recombinant plasmids
• This results in the cloning of many hummingbird DNA
fragments, including the β-globin gene
Fig. 20-4-1
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Bacterial
plasmid
Sticky
ends
Gene of interest
Hummingbird
DNA fragments
Fig. 20-4-2
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Fig. 20-4-3
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
Fig. 20-4-4
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
Restriction
site
ampR gene
Sticky
ends
Bacterial
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying recombinant
plasmid with disrupted lacZ gene
One of many
bacterial
clones
Screening a Library for Clones Carrying a
Gene of Interest
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid hybridization
• A probe can be synthesized that is complementary
to the gene of interest
• For example, if the desired gene is
5 … G G C T A A C T T A G C … 3
– Then we would synthesize this probe
3 C C G A T T G A A T C G 5
• The DNA probe can be used to screen a large
number of clones simultaneously for the gene of
interest
• Once identified, the clone carrying the gene of
interest can be cultured
Fig. 20-7
TECHNIQUE
Radioactively
labeled probe
molecules
Multiwell plates
holding library
clones
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
•
Nylon membrane
Nylon
Location of
membrane
DNA with the
complementary
sequence
Amplifying DNA in Vitro: The Polymerase
Chain Reaction (PCR)
• The polymerase chain reaction, PCR, can produce
many copies of a specific target segment of DNA
• A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
Fig. 20-8
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1 Denaturation
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Fig. 20-8a
5
TECHNIQUE
3
Target
sequence
Genomic DNA
3
5
Fig. 20-8b
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Fig. 20-8c
Cycle 2
yields
4
molecules
Fig. 20-8d
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Concept 20.2: DNA technology allows us
to study the sequence, expression, and
function of a gene
• DNA cloning allows researchers to
• Compare genes and alleles between individuals
• Locate gene expression in a body
• Determine the role of a gene in an organism
• Several techniques are used to analyze the DNA of
genes
Gel Electrophoresis and Southern Blotting
• One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis
• This technique uses a gel as a molecular sieve to
separate nucleic acids or proteins by size
• A current is applied that causes charged molecules
to move through the gel
• Molecules are sorted into “bands” by their size
Video: Biotechnology Lab
Fig. 20-9
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Power
source
– Cathode
Anode
+
Gel
1
Power
source
–
+
Longer
molecules
2
RESULTS
Shorter
molecules
Fig. 20-9a
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Power
source
Anode
– Cathode
+
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
Fig. 20-9b
RESULTS
• In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a DNA
molecule are sorted by gel electrophoresis
• Restriction fragment analysis is useful for comparing
two different DNA molecules, such as two alleles for
a gene
• The procedure is also used to prepare pure samples
of individual fragments
Fig. 20-10
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
DdeI
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles