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Recombinant DNA Technology
Methods for Isolating,
Amplifying and Studying
Specific DNA Sequences
Cutting and
Joining DNA
Fragments
Restriction
Enzyme:
cleaves DNA
at specific
sequences
DNA Ligase:
joins DNA
fragments by
sealing nicks
in backbone
Use of the Restriction Enzyme Bam H1
5’— G G A T C C — 3’
3’— C C T A G G — 5’
Results in
5’— G
3’— C C T A G
sticky end
G A T C C — 3’
G — 5’
sticky end
DNA fragments with complementary sticky ends
can associate to form new DNA combinations.
Viewing DNA Fragments:
Agarose Gel Electrophoresis
Locating DNA Fragments by
Southern Blotting
Probe is
complementary to
DNA of interest
DNA Fragments
are transferred
to membrane
A subset of
fragments
binds to the
probe
Cloning Genes
• Gene cloning = isolation of a specific
segment of DNA in a form that can be
duplicated in another cell
• Requires a
– Vector = carrier of DNA segment
– Host = cell in which foreign DNA can be
duplicated and its gene product may be
synthesized
• Recombinant DNA = DNA produced by
joining fragments from two or more
different sources
Cloning Vectors
• For Bacterial Hosts
– Plasmid
ampR
Bam H1
tetR
ampR
tetR gene disrupted, no longer functional
Cloning Vectors
• For Bacterial Hosts
– Bacteriophage
– Cosmid
– Expression Vector
Cloning Vectors
• For Eukaryotic Hosts
– Shuttle Vector
– YAC = yeast artificial chromosome
– Ti plasmid
Tools for Finding Genes
• DNA library = collection of clones from
one DNA donor
– Categorized by: vector, source of
DNA
– Genomic library = all of the
sequences from the genome of a
single organism
– cDNA library= complementary DNA,
made using mRNA as a template
Producing a Genomic Library
Source of DNA for
Library
Many recombinant
molecules are formed,
each with a different
DNA insert
Producing cDNA
Primer binds to
poly-A tail
DNA produced from
RNA template
Partial digestion of
RNA template
Second DNA strand
produced using
remaining RNA
segments as primers
Gaps in backbone
sealed
Tools for Finding Genes
Probe: sequence of DNA or RNA that
is complementary to the gene of
interest; Used to locate a copy of the
gene in a library by hybridization
AATGCCAT
TTACGGTA
Denature DNA by heating
Add Probe
Probe Binds to gene
AATGCCAT
TTACGGTA
TTACGGTA
Screening a Library with a Probe
Amplifying DNA with the
Polymerase Chain Reaction (PCR)
• Copies a specific region in the DNA
• Can be used with small amounts of
sample
• Uses repeated cycles of heating to
denature DNA and cooling to synthesize
new DNA
• Requires
– primers to begin synthesis
– heat-stable polymerase (Taq polymerase)
Polymerase
Chain Reaction
DNA Sequencing by
the Sanger Method
Relies on Chain Termination during
replication due to incorporation of
Dideoxynucleotides
OH H
H
H
deoxyribose dideoxyribose
Replicate unknown DNA using dideoxynucleotides
to terminate growing chains at various positions
Unknown Sequence
3’-A-G-C-C-C-A-G-A-T-T-5’
Complementary Products
Black= dideoxynucleotide
5’-T-3’
5’-T-C-G-G-G-3’
5’-T-C-G-3’
5’-T-C-G-G-G-T-C-T-3’
5’-T-C-3’
5’-T-C-G-G-G-T-C-T-A-3’
5’-T-C-G-G-G-T-C-T-A-A-3’
5’-T-C-G-G-G-T-C-3’
5’-T-C-G-G-G-T-3’
5’-T-C-G-G-3’
Use gel electrophoresis to separate replicated
fragments that differ by one base
A
Dideoxynucleotide
T
C
G
Size of DNA on gel shows distance from start of replication
Labeling of dideoxynucleotide shows complementary
sequence
Determine unknown sequence from complementary
sequence with base-pairing rules
A
Dideoxynucleotide
T
C
G
Complementary
Sequence
5’-T-C-G-G-G-T-C-T-A-A
5’-T-C-G-G-G-T-C-T-A
5’-T-C-G-G-G-T-C-T
5’-T-C-G-G-G-T-C
5’-T-C-G-G-G-T
5’-T-C-G-G-G
5’-T-C-G-G
5’-T-C-G
5’-T-C
5’-T
Complementary Sequence
Unknown Sequence
Applications of Recombinant
DNA Technology
Genetic Testing and Gene Therapies
Products of Biotechnology
RFLP Analysis
• RFLP = Restriction
Fragment Length
Polymorphism
• Identifies differences in
the length of restriction
fragments derived from
similar DNA sequences
• Analyzed by Southern
Blotting
• Used in gene mapping
RFLP Analysis in Paternity Testing
RFLP Analysis in Forensics
1 2 3
Suspects
CS
RC
IE
MN
EE
4 5 67
Suspects
DNA Fingerprinting
• Detects patterns for short repeated
sequences that are highly variable among
humans
• Described as
– VNTR: Variable
Number of Tandem
Repeats
– STR: Short Tandem
Repeats
– Microsatellites
Analysis using Microsatellite sequences
Genetic Testing
Gene
Therapy
Andrew Gobea was born with SCID, an immune
deficiency due to a defect in the ADA gene.
Four days after his birth, he received an injection
of his own stem cells carrying functional ADA genes.
Andrew’s immune system began producing ADA,
but the amount was not sufficient to maintain full
health. He needs daily injections of ADA enzyme.
RNA Interference as a Possible
Treatment for High Cholesterol
• High levels of ApoB, a protein
component of blood lipid carriers, are
associated with high cholesterol levels
• In monkeys, siRNA for ApoB has
lowered the mRNA for ApoB in liver cells
and also reduced serum cholesterol
levels
siRNA for ApoB
was delivered in
lipid capsules
Recombinant DNA Products
Pharmaceutical
Used for
Factor VIII
Blood Clotting
Human Growth
Hormone
Pituitary Dwarfism
Insulin
Diabetes
Interferon
Cancer
Tissue Plasminogen
Activator
Vaccine
Heart Attack
Hepatitis B
Genetically Modified
Organisms
 Introducing or modifying
specific genes to alter the
phenotype of an organism
 Transgenic: organism that
contains a gene from another
species in all of its cells
 Transgenic Animals
 Models of Human Disease
 Produce Pharmaceuticals
Studying Gene
Function with
Knockout Mice
• neo+ gene gives
resistance to G418
• tk+ gene gives
sensitivity to
gancyclovir
• Insertion of neo+
into target gene
yields neo+ and tkcells, resistant to
G418 and to
gancyclovir
Transgenic
Plants
“Golden” rice with
beta-carotene and
extra iron
Round Up Ready Soybeans
are resistant to herbicide
Bt Corn produces
its own pesticide
What Are Some Concerns?