Recombinant DNA
Chapter 18
Learning Objectives
• Define Clone and DNA Cloning
• List the three steps of production of recombinant
DNA
• Describe the characteristics and uses of a
restriction endonuclease
• Diagram the process of identifying a transformed
bacterial colony containing a gene of interest
using Ampicillin Resistance, Lactose Metabolism
plasmids and nucleic acid hybridization probes
Learning Objectives
• Explain the uses of RFLPs
• Describe the process of producing a
transgenic organism, and explain its
usefulness
DNA cloning
• Clone: genetically identical cells or
individuals derived from a single ancestor
• DNA cloning: a method of producing a
large amount of the DNA of interest
• Large amounts of identical pieces of DNA
enable us to manipulate and recombine
genetic material
DNA Technologies
• DNA technologies are used in molecular testing
for many human genetic diseases
• DNA fingerprinting used to identify human
individuals and individuals of other species
• Genetic engineering uses DNA technologies to
alter the genes of a cell or organism
• DNA technologies and genetic engineering are a
subject of public concern
Recombinant DNA
• DNA from two or more sources joined
together
• DNA of interest can be spliced into
bacterial plasmids (recombination)
• Plasmids replicate (amplification)
• Plasmids (DNA) are extracted (isolation)
Endonucleases
• Restriction enzymes (endunucleases) cut
DNA at specific sequences in restriction
sites
– Restriction fragments result
– Sticky ends have unpaired bases at cuts
which will hydrogen bond
– Ligase stitches together paired sticky ends
Restriction site
for EcoRI
DNA
1 EcoRI restriction enzyme
cleaves sugar–phosphate
backbones at arrows.
Sticky end
Sticky end
Another DNA fragment
produced by EcoRI digestion
2 DNA fragments with the same
sticky ends can pair. Shown here
is a DNA fragment inserting
between two other DNA
fragments, as happens when
inserting a DNA fragment into a
bacterial plasmid.
Nick in sugar–phosphate backbone
3 Nicks in sugar–
phosphate backbones
are sealed by DNA ligase.
Recombinant DNA molecule
Fig. 18-3, p. 374
Recombinant DNA
• Restriction endonucleases
• Each type is specific for a four to eight
base pair long palindromic recognition
sequence of DNA
• Palindrome- reads the same on each
strand 3’ to 5’ like GAATTC
CTTAAG
Gene of
interest
Cell
DNA fragments with
sticky ends
Restriction
site
ampR
gene
lacZ+
gene
Plasmid
cloning
vector
Cut plasmid cloning vectors
with a restriction
enzyme to produce sticky ends
Fig. 18-4a, p. 375
Inserted genomic
DNA fragment
Recombinant
DNA molecules
4 Introduce recombinant molecules
into bacterial cells; each bacterium
receives a different plasmid. As the
bacteria grow and divide, the
recombinant plasmids replicate,
thereby amplifying the piece of DNA
inserted into the plasmid.
Bacterium
Bacterial
chromosome
Progeny
bacteria
5 Identify the bacterium containing
the plasmid with the gene of interest
inserted into it. Grow that bacterium
in culture to produce large amounts
of the plasmid for experiments with
the gene of interest.
Fig. 18-2b, p. 373
Recombinant DNA
• Break cells and use restriction enzyme to isolate
DNA of interest (prokaryotic or eukaryotic)
• Insert into plasmid (recombination)
• Transform into bacteria (replication)
• Not very efficient, so for the third step (isolation)you need to have engineered a way to find the
bacteria of interest
Four possibilities
1. Desired outcome: plasmid, lac+ broken,
gene of interest inserted
2. Bacteria transformed with plasmid, but
wrong gene inserted
3. Bacteria transformed with plasmid onlyno gene at all inserted
4. Bacteria not transformed
Inserted DNA
fragments with
gene of interest
Resealed plasmid
Inserted DNA
fragment without cloning vector with no
gene of interest inserted DNA fragment
Recombinant plasmids
Nonrecombinant plasmid
Fig. 18-4b, p. 375
Recombinant DNA
• Insert into special screening plasmid- which
contains the same restriction enzyme site used
above, located in a lacZ gene.
• For recombination screening the lacZ gene is
broken successfully, it will be white. If not, it will
be blue.
• The plasmid also contain ampicillin resistance
• If transformation worked, the bacteria will grow
on plates containing ampicillin. Those who were
not transformed will not grow.
Bacteria transformed with plasmids
Selection:
Transformed bacteria grow
on medium containing
ampicillin because of ampR
gene on plasmid.
Screening:
Blue colony contains
bacteria with a nonrecombinant plasmid;
that is, the lacZ+
gene is intact.
White colony contains bacteria
with a recombinant plasmid;
that is, the vector with an
inserted DNA fragment. Once
the white colony with the gene
of interest is identified, it can
be grown in culture to produce
large quantities of the plasmid.
Bacteria not
transformed
with a plasmid
Untransformed
bacterium
cannot grow on medium
containing ampicillin.
Plate containing
ampicillin and X-gal
Fig. 18-4c, p. 375
DNA Hybridization
• Uses nucleic acid probe to identify gene of
interest in set of clones
– Probe has tag for detection
– Identified colony produces large quantities of
cloned gene
Culture medium
containing ampicillin
Bacterial
colony
Filter paper
Replica of bacterial
colonies
Filter paper
Fig. 18-5a, p. 377
Labeled probe
Plasmid DNA
(single stranded) (single stranded)
Labeled singlestranded DNA
probe for the
gene of interest
Bag
Filter
Hybridization has occurred
between the labeled probe and
the plasmids released from the
bacteria in this colony. The
hybridization is detected in
subsequent steps.
Fig. 18-5b, p. 377
Developed
photographic
film
Corresponds to
one colony on
master plate
Original master plate
Fig. 18-5c, p. 377
4 Possibilities
Outcome
AMP
LAC
PROBE
Right Gene
yes
No
Yes
Wrong Gene
Yes
No
No
Plasmid only
Yes
Yes
n/a
No Plasmid
No
No
n/a
How else do we use
Restriction
Endonuclease?
RFLPs
• Restriction fragment length polymorphisms
– DNA sequence length changes due to varying
restriction sites from same region of genome
– Sickle cell anemia has RFLPs
• Southern blot analysis uses
electrophoresis, blot transfer, and labeled
probes to identify RFLPs
– Alternative is PCR and electrophoresis
β-Globin gene
175 bp
201 bp
Normal
allele
MstII
MstII
MstII
376 bp
Sickle-cell
mutant allele
MstII
MstII
Region of probe
used to screen for
sickle-cell mutation
Fig. 18-8, p. 381
DNA Fingerprinting
• Distinguishes between individuals
– Uses PCR at multiple loci within genome
– Each locus heterozygous or homzygous for
short tandem repeats (STR)
• PCR amplifies DNA from STR
– Number of gel electrophoresis bands shows
amplified STR alleles
– 13 loci commonly used in human DNA
fingerprinting
Forensics and Ancestry
• Forensics compares DNA fingerprint from
sample to suspect or victim
– Usually reported as probability DNA came
from random individual
• Common alleles between children and
parents used in paternity tests
– Same principle used to determine
evolutionary relationships between species
a. Alleles at an STR locus
STR locus
Left PCR primer
DNA
9 repeats
Right PCR primer
3 different
alleles
11 repeats
15 repeats
Fig. 18-10a, p. 383
b. DNA fingerprint analysis of the STR locus by PCR
B
A
C
Cells of
three
individuals
Extract genomic DNA
and use specific
primers to amplify
the STR locus using
the PCR.
Anyalyze PCR product by gel electrophoresis
Positions
corresponding to
alleles of STR locus
15
11
9
A
B
C
11,11 15,9 11,9
Fig. 18-10b, p. 383
Genetic Engineering
• Transgenic organisms
– Modified to contain genes from external
source
• Expression vector has promoter in plasmid
for production of transgenic proteins in E.
coli
– Example: Insulin
– Protocols to reduce risk of escape
Animal Genetic Engineering
• Transgenic animals used in research,
correcting genetic disorders, and protein
production
• Germ-line cell transgenes can be passed
to offspring (somatic can not)
– Embryonic germ-line cells cultured in quantity,
made into sperm or eggs
– Stem cells
Germ-line cells derived
from mouse embryo
Transgene
Cell with
transgene
Pure population of
transgenic cells
Fig. 18-11a, p. 385
Mice have transgenic cells in
body regions including germ line
Genetically engineered
offspring—all cells transgenic
Fig. 18-11b, p. 385
Gene Therapy
Attempts to correct genetic disorders
– Germ-line gene therapy can’t be used on
humans
– Somatic gene therapy used in humans
• Mixed results in humans
– Successes for adenosine deaminase
deficiency (bubble kid) and sickle-cell
– Deaths from immune response and leukemialike conditions
– http://history.nih.gov/exhibits/genetics/sect4.htm
Animal
Genetic Engineering
•
“Pharm”
animals produce proteins for humans
– Usually produced in milk for harmless extraction
• Cloned mammals produced by implantation of diploid
cell fused with denucleated egg cell
– Low cloning success rate
– Increased health defects in clones
– Gene expression regulation abnormal
Learning Objectives
• Define Clone and DNA Cloning
• List the three steps of production of recombinant
DNA
• Describe the characteristics and uses of a
restriction endonuclease
• Diagram the process of identifying a transformed
bacterial colony containing a gene of interest
using Ampicillin Resistance, Lactose Metabolism
plasmids and nucleic acid hybridization probes
Learning Objectives
• Explain the uses of RFLPs
• Describe the process of producing a
transgenic organism, and explain its
usefulness