DNA Technology
• Genetic Engineering – direct manipulation of
genes for practical purposes
– Scientists can make recombinant DNA and
then introduce it into cultured cells that
replicate the DNA and may express its
genes, yielding a desired protein.
– Often, E. coli is used as the “host”
• Biotechnology – manipulations of organisms
to make useful products.
• Recombinant DNA – genes from two different
sources are combined in vitro (outside the
living body in a lab).
Techniques for Gene Cloning
Techniques for Gene Cloning
•
Plasmid – circular DNA that replicates within
bacterial cells (separate from bacterial
chromosomes)
Isolation of plasmid
Gene insertion into plasmid
Plasmid put into bacterial cell
Cell cloned
Identification of desired clone
Copies of the gene/Copies of the protein
1.
2.
3.
4.
5.
6.
•
Used in gene therapy (HGH) and agriculture
(resistance to pests)
Restriction Enzymes
• Restriction enzymes cut up foreign DNA –
recognize short nucleotide sequences
• Restriction site – area recognized by
restriction enzyme (5’ – 3’)
• Restriction fragments – nucleotide
sequence left after DNA has been cut.
• Sticky end – single-stranded DNA
fragment; DNA ligase can permanently fuse
fragments together
Restriction
Enzymes
Transgenic organism
An organism that contains
genes from other species.
Results from the formation of
recombinant DNA.
Ex: Fruit fly w/firefly gene for
luciferase
Bacterial Transformation
•Another method used to form
transgenic organisms
•Plasmids inserted to change
the proteins produced by cells
•Lab 6A
•pGLO
DNA
Cloning
DNA Cloning
•
•
•
Cloning Vector – the original bacterial
plasmid
Procedure: Cloning a Eukaryotic Gene in
a Bacterial Plasmid
The E. coli plasmid has two useful genes:
ampR (resistant to ampicillian) and lacZ
(catalyses hydrolysis of lactose)
1. Isolation of vector (bacterial plasmid) and
gene-source DNA (human tissue)
DNA Cloning
2. Insertion of DNA into the vector
•
•
•
•
•
Restriction enzyme cuts plasmid DNA at single
restriction site
Cuts human DNA making thousands of
fragments
One of these fragments carries the gene we
want
The restriction enzyme creates compatible sticky
ends on both the human DNA fragments and the
plasmid DNA
The DNA and plasmids are combined – DNA
ligase “glues” the pieces together
DNA Cloning
3. Introduction of cloning vector into cells
•
•
Transformation – uptake of naked DNA from
surrounding solution
Some acquire the desired DNA; others take up
other DNA
4. Cloning of cells
•
•
Put bacterial cells on a plate containing medium
with ampicillin and X-gal
Bacteria with recombinant plasmids carrying
foreign DNA with form white colonies on medium
containing ampicillin and X-gal
DNA Cloning
5. Identification of cell colonies carrying the gene
of interest
• Nucleic acid hybridization – base pairing
between the gene and a complementary
sequence using a nucleic acid probe
• The probe can be labeled with a radioactive
isotope or a fluorescent tag
• Then the strand of DNA is separated by
denaturing it with heat or chemicals
• The desired DNA can then be isolated an
grown in large amounts
Cloned Genes Are Stored
in DNA Libraries
• A complete set
of thousands of
recombinant
plasmid clones
• Can be stored
as a plasmid
library (bacterial
cells) or phage
libraries
(viruses)
Polymerase Chain Reaction
(PCR) Clones DNA In Vitro
• Any piece of DNA can be quickly copied many
times without using a host cell
• DNA is placed in a test tube with:
– Special DNA polymerases (first isolated from
bacteria growing in hot springs – the enzyme does
not denature with heat)
– Supply of nucleotides
– Synthetic single-stranded DNA that serves as
primers
• Can be used to amplify a specific gene prior to
cloning
• PCR can be used
to quickly amplify
DNA from a
40,000 year old
woolly mammoth;
tiny amounts of
blood, tissue, or
semen from a
crime scene; and
DNA of viral
genes from HIVinfected cells
DNA Analysis and Genomics
•
1.
2.
3.
4.
Suppose we have cloned a DNA segment
carrying a human gene of interest – now we
begin to ask some far-reaching questions:
Do genes differ in different people, and are
certain alleles associated with a hereditary
disorder?
Where and when is it expressed?
What is its location within the genome?
Evolutionary questions - Species to species
differentiation?
DNA Analysis and Genomics
• Genomics – the study of whole sets of genes
and their interactions
• Gel Electrophoresis – method of sorting DNA
molecules into bands, each consisting of
molecules of the same length
– Separation on the basis of size, electrical charge,
and other physical properties
• Restriction fragments – detect DNA
differences that affect restriction sites
– Gel Electrophoresis used to sort DNA fragments
by size after being treated with restriction
enzymes
Gel Electrophoresis
1. Digest two different DNA samples with
same restriction enzyme (the samples
should differ in one or more restriction
site)
2. Use nucleic acid hybridization with a
specific probe to label discrete bands
that derive from our gene of interest
(usually used in combination with
Southern blotting)
Gel Electrophoresis
Gel Electrophoresis
Gel Electrophoresis
The DNA fingerprints below represent four different
individuals.
Which of the following
statements is consistent with
the results?
• B is the child of A and C.
• C is the child of A and B
Which of the following are
probably siblings?
• A and D
• C and D
Entire Genomes Can Be
Mapped at the DNA Level
• Human Genome
Project – begun in
1990 to map all
the human genes
• Surprisingly, there
are few genes in
the Human
Genome
Practical Application of DNA Technology
• Diagnosis of Diseases
– PCR can be used to amplify and detect HIV DNA
in blood or tissue samples
– Scientists can diagnose hundreds of human
genetic disorders before the onset of symptoms,
even before birth
– We can identify symptomless carriers of
potentially harmful recessive alleles
– Genes have been cloned for many human
diseases, including hemophilia, cystic fibrosis, and
Duchenne muscular dystrophy
Practical Application of DNA Technology
• Human Gene Therapy – the alteration of an
afflicted individual’s genes
– Theoretically, it is possible to replace or
supplement the defective gene with a normal
allele
– The new allele could be inserted into the somatic
cells of the tissue affected by the disorder
– The cells that receive the normal allele must be
ones that will continue to reproduce during the
patient’s life – one example is stem cells from
bone marrow that give rise to all blood and
immune cells
Practical Application of DNA Technology
• Use of stem cells
to promote
healthy bone
marrow
Practical Application of DNA Technology
• Pharmaceutical Products
– Mostly proteins
– Human insulin
– Human growth hormone (HGH)
– Tissue plasminogen activator (TPA) – protein that
helps dissolve blood clots (very expensive)
– Drugs that mimic a receptor protein that HIV binds
to in entering white blood cells – the HIV binds to
the drugs and fails to enter the blood cell
– Vaccines
Practical Application of DNA
Technology
• Reproductive Cloning
– Producing complete, genetically identical
animals
– Usually talked about in the press
• DOLLY the SHEEP.
Practical Application of DNA Technology
• Forensic Uses
– DNA fingerprinting
• Environment Uses
– Sewage treatment plants rely on the ability of
microbes (bacteria and protists, mainly) to
degrade many organic compounds into nontoxic
forms
• Agricultural Uses
– Animal Husbandry and “Pharm” Animals
– Transgenic organisms – their genomes carry
genes from another specie
Practical Application of DNA Technology
• Agricultural Uses (continued)
– Genetic engineering in plants (editable cotton
seed – 2006)
– Delayed ripening
– Resistance to spoilage and disease
– Increase nutritional value (“golden rice”)
SO THE BIG QUESTION IS: