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DNA TECHNOLOGY
DNA recombination or genetic
engineering is the direct manipulation
of genes for practical purposes
Recombinant DNA technology
• Refers to the set of techniques for
combining genes from different sources in
vitro( in a test tube) and transfering this DNA
into a cell so it can be expressed.
• These techniques were first developed
around 1975 and resulted in the appearance
of the Biotechnology industry
CONNECTION
•12.7 DNA technology is changing the
pharmaceutical industry
– DNA technology
•
Is widely used to produce medicines and to
diagnose diseases
What is biotechnology?
• The use of living organisms to do practical
tasks.
• Examples:
• The use of microorganisms to make cheese
and wine
• Selective breeding of livestock and crops
• Production of antibiotics from
microorganisms
• Production of monoclonal antibodies
What is the goal of biotechnology?
• To find practical applications of DNA
tecniques for the improvement of human
health and food production
• Examples:
Making gene products using Genetic Engineering
Uses in basic research
Medical uses. Diagnosis of disease
Making vaccines and other pharmaceutical products
Forensic uses of DNA such as DNA fingerprinting
Agricultural uses such as manipulatingplant genes and
making transgenic plants.
The tools of recombinant DNA
•
•
•
•
Plasmids
Restriction enzymes
Gel electrophoresis
PCR ( polymerase chain reaction)
BACTERIAL PLASMIDS AND
GENE CLONING
•12.1 Plasmids are used to customize bacteria:
An overview
– Gene cloning is one application of DNA
technology
•
Methods for studying and manipulating
genetic material
– Researchers can insert desired genes into
plasmids, creating recombinant DNA
•
And insert those plasmids into bacteria
Bacterium
Cell containing gene
of interest
1 Plasmid
isolated
2 DNA
isolated
3 Gene inserted
into plasmid
Plasmid
Bacterial
chromosome
Recombinant DNA
(plasmid)
DNA
Gene of
4 Plasmid put into interest
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Figure 12.1
Copies of protein
Clone of cells
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
•Therapeutic hormones
– In 1982, humulin, human insulin produced by
bacteria
•
Became the first recombinant drug approved
by the Food and Drug Administration
Figure 12.7A
Gene therapy may someday help treat a
variety of diseases
– Gene therapy
•
Is the alteration of an afflicted individual’s genes
Cloned gene
(normal allele)
1 Insert normal gene
into virus
Viral nucleic
acid
Retrovirus
2 Infect bone marrow
cell with virus
3 Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
Figure 12.13
4 Inject cells
into patient
•12.11 Restriction fragment length
polymorphisms can be used to detect
differences in DNA sequences
•How Restriction Fragments Reflect DNA
Sequence
– Restriction fragment length polymorphisms (RFLPs)
•
Reflect differences in the sequences of DNA samples
Crime scene
Suspect
w
Cut
C
C
G
G
G
G
C
C
z
A
C
G
G
T
G
C
C
C
C
G
G
G
G
C
C
x
Cut
y
Figure 12.11A
C
C
G
G
G
G
C
C
Cut
y
DNA from chromosomes
CONNECTION
•12.9 DNA microarrays test for the expression
of many genes at once
– DNA microarray assays
•
Can reveal patterns of gene expression in
different kinds of cells
– DNA microarray
DNA microarray
Each well contains
DNA
from a particular gene
Actual size
(6,400 genes)
1 mRNA
isolated
Reverse transcriptase
and fluorescent DNA
nucleotides
2 cDNA made
from mRNA
4 Unbound
cDNA rinsed
away
Fluorescent
spot
3 cDNA applied
to wells
Nonfluorescent
spot
cDNA
DNA of an
expressed gene
Figure 12.9
DNA of an
unexpressed gene
•12.10 Gel electrophoresis sorts DNA
molecules by size
Mixture of DNA
molecules of
different sizes
–
–
Longer
molecules
Power
source
Gel
+
Shorter
molecules
+
Figure 12.10
Completed gel
– After digestion by restriction enzymes
•
The fragments are run through a gel
1
–
2
Longer
fragments
z
x
w
Shorter
fragments
Figure 12.11B
+
y
y
•12.11 Restriction fragment length
polymorphisms can be used to detect
differences in DNA sequences
•Using DNA Probes to Detect Harmful Alleles
– Radioactive probes
•
Can reveal DNA bands of interest on a gel
– Detecting a harmful allele using restriction
fragment analysis
1 Restriction fragment
preparation
I
II
III
Restriction
fragments
2 Gel electrophoresis
I II III
3 Blotting
Filter paper
4 Radioactive probe
Radioactive, singlestranded DNA (probe)
Probe
5 Detection of radioactivity
(autoradiography)
I
II
III
Film
Figure 12.11C
I
II
III
CONNECTION
DNA technology is used in courts of law
•DNA and Crime Scene Investigations
– Many violent crimes go unsolved
•
For lack of enough evidence
– If biological fluids are left at a crime scene
•
DNA can be isolated from them
– DNA fingerprinting is a set of laboratory procedures
•
•
That determines with near certainty whether two
samples of DNA are from the same individual
That has provided a powerful tool for crime scene
investigators
Investigator at one
of the crime scenes
(above), Narborough,
England (left)
DNA Fingerprinting
1st-The DNA molecule is cut with restriction enzymes
2nd- we have to separate the fragments
This is done by a technique called gel electrophoresis
The DNA is placed on a tray filled with gel through which an
electric current runs causing the fragments to move
through the gel. The segments separate by how far they
move in the gel according to size.
The DNA will form bands corresponding to the bases (and no
two people have the same sequence of bases) in the gel which
are unique for each individual. This is DNA fingerprinting
– DNA fingerprinting can help solve crimes
Defendant’s
blood
Blood from
defendant’s clothes
Figure 12.12A
Victim’s
blood
Figure 12.12B
Safety and ethical issues
• Methods for purifying the DNA
• Vectors for carrying the DNA into cells and
replicating it
• Techniques for determining nucleotide
sequences of DNA molecules