Download DNA TECHNOLOGY AND GENOMICS

DNA TECHNOLOGY
the new genetics
Chapter 13
Ch 13 VOCABULARY
put a + by the terms you know and – by the ones you don’t.
GMO
Clone
Vaccine
biotechnology
Genetic
engineering
GFP
ligation
plasmid
endonuclease
technology
PCR
insulin
Gene
expression
Transgenic
Gel
electrophoresis
DNA
Gene
therapy
Restriction
enzyme
Somatic cell
DNA Fingerprint
Nuclear transfer
HGH
Dolly
vector
Biomedical
agriculture
RFLP
interleukin
ethical
interferon
transformation
GENERATE YOUR OWN QUESTIONS:
1pt question words: Who? What? Where? When?
2pt question words: Which? How?
3pt question words: Why?
Transgenic/Recombinant organisms contain
DNA that was not part of their original
genome.
Green fluorescent
protein (GFP) is
responsible for the
green bioluminescence
of the jellyfish Aequorea
victoria.
This is a GM mouse!
5. The genetic composition of cells can be altered by
incorporation of exogenous DNA into the cells. As a
basis for understanding this concept:
a.Students know the general structures and functions of
DNA, RNA, and protein.
b. Students know how to apply base-pairing rules to
explain precise copying of DNA during
semiconservative replication and transcription of
information from DNA into mRNA.
5. The genetic composition of cells can be altered by
incorporation of exogenous DNA into the cells. As a
basis for understanding this concept:
c. Students know how genetic engineering (biotechnology) is used to
produce novel biomedical and agricultural products.
d.* Students know how basic DNA technology (restriction digestion
by endonucleases, gel electrophoresis, ligation, and
transformation) is used to construct recombinant DNA molecules.
e.* Students know how exogenous DNA can be inserted into
bacterial cells to alter their genetic makeup and support expression
of new protein products.
Genetic Engineering is the application of
molecular genetics for practical purposes.
Uses:
1) Identify genes for specific traits
2) Transfer genes for a specific trait from one
organism to another.
Tools for manipulating genes:
1) Restriction enzymes (endonucleases)
2) Cloning vector (bacterial plasmid)
The Human Genome Project
Goals of the Human Genome Project:
•
•
•
Determine the nucleotide sequence of the entire
human genome.
Map the location of every gene on each
chromosome.
Compare the genomes of other organisms to the
human genome to understand:
1. How genomes are organized.
2. How gene expression is controlled.
3. How cellular growth and differentiation are
under genetic control.
4. How evolution occurs.
GENOMIC LIBRARY
is a catalog of the DNA of a species
• Cut up the DNA of the species into tiny
pieces using restriction enzyme.
• Put each DNA fragment into a different
cloning vector- ex. plasmid.
• Put each recombinant plasmid into a
separate bacterium.
• FREEZE until needed.
Gene Therapy
• Treating a genetic disorder by introducing
a gene into a cell or by correcting a gen
defect in a cell’s genome.
• 1990’s.
• Cystic Fibrosis, AIDS, Ovarian Cancer.
Practical Uses of DNA
Technology:
• Pharmaceuticals- HGH, Interferons,
Interleukins etc.
• Vaccines- solution that contains a
harmless version of a virus or bacterium
to stimulate an immune response &
formation of “memory” cells.
• Increased Agricultural Yields- ex. crops
that don’t need fertilizer.
Ethical Issues
• Describe two potential safety and
environmental problems that could
result from genetic engineering.
DNA technology can be used to:
1. Cure diseases
2. Treat genetic disorders
3. Improve food crops
Golden rice contains beta-carotene, which our bodies use to make vitamin A.
Figure 20.18 “Pharm” animals secrete spider silk in their milk
Could use this
Technology to
Make insulin or
Human growth Hormone
Figure 20.16 One type of gene therapy procedure
transgenic organism
•
•
Contains new DNA.
Ex. Bacterium with plasmid containing
insulin gene.
•
•
Grow bacteria (beaker or petri dish)
Bacteria express (transcribe/translate) the
cloned gene to make insulin.
Insulin is extracted (purified) from the
medium.
Treatment for Diabetes.
•
•
Injecting DNA into an embryo…
how you create a “cloned organism”
SCNT Somatic Cell Nuclear Transfer
Tools for manipulating genes
Restriction Enzymes
(endonucleases)
• Molecular “scissors”
that cut DNA at
specific sequences.
• Provide protection
for bacteria against
viruses.
3 examples of restriction enzymes:EcoRI, BamHI, HindIII
Restriction Enzymes or
Restriction Endonucleases
• Are bacterial enzymes that cut DNA
molecules into smaller pieces
• They cut the DNA at a specific site, a known
sequence of DNA .
for example: CTTAAG
GAATTC
• EcoRI cuts between the G and A, leaving two
open ends with single-chain “tails” called
“sticky ends”
Ex. CTTAA
and
G
G
AATTC
• Sticky ends readily bind to
complementary chains of DNA.
• Thus, pieces of DNA that have been cut
with the same restriction enzyme can
bind together to form a new sequence
of nucleotides.
Restriction Enzymes
CLONING VECTORS
• Restriction enzymes can be used to isolate a specific
gene of interest from a donor called the donor gene.
• A plasmid is a ring of DNA found in a bacterium in
addition to its main chromosome.
• Cut the plasmid with the same restriction enzyme as the
donor gene to “splice” it into the plasmid.
• Insert this recombinant DNA plasmid into the
bacterium.
• When the bacteria reproduces by binary fission the
recombinant plasmid does too, we call this cloning a
gene.
• When a virus is used as the vector for gene
transmission, this is called transduction.
Plasmids: small circular
pieces of DNA.
Plasmids often contain
genes for
antibiotic
resistance.
Conjugation- when bacteria exchange plasmids.
Transformation:
• bacteria can
incorporate new
DNA into their
genome.
• They do it all the
time naturally.
• Pick up plasmids
from their
environment.
Transplanting Genes
• Plasmids are used to transfer a gene to
bacteria so the bacteria will produce a
specific protein.
• Ex. Human insulin
Human Growth Hormone
Just give the bacteria food and they will
reproduce and produce your protein. You
have a protein factory!!!! (clean up your
protein, separate it and purify it)
1. Plasmid & Donor gene
(cut by same RE)
2. Spliced w/ ligase
3. Recombinant plasmid
inserted into bacteria
(transformed)
4. Bacteria replicates &
produces
5. Gene clone- exact
copy of the gene.
Cloning A Gene
Recombinant Organisms
• Organisms that receive
the recombinant
plasmid.
• Ex. Glo fish, glowing cat,
pharm animals, golden
rice, roundup ready
soybeans,
How to create a recombinant
plasmid
1) Treat plasmid and
donor gene with the
same restriction
endonuclease (they
used EcoRI)
2) This creates the same
sticky ends on
plasmid and donor
gene DNA.
3) Place both together with DNA ligase to join the donor
gene with the plasmid.
Phosphodiester bonds link sugar-phosphates of
nucleotides… hydrogen bonds form/break
spontaneously.
Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants
DNA Technology Techniques
PCR
Gel Electrophoresis
Polymerase Chain Reaction
a way to make millions of copies of DNA!!!
What you need:
• DNA sample
• Free nucleotides
– A heat resistant DNA
polymerase
– Example: Taq polymerase
• Primers: short segments(2030bases) of DNA
complementary to the ends of
the DNA being copied.
Polymerase Chain Reaction
1) Denature the original
strand of DNA with heat.
1)
Cool the mixture, allowing
the primers to bind
(anneal) to the DNA.
1)
The DNA polymerase
binds free nucleotides to
the primer using the
original DNA strand as a
template. This creates two
copies of the DNA sample.
1)
Repeat.
Gel Electrophoresis
• Technique used to separate
restriction fragments.
• DNA fragments of different
lengths are separated as they
diffuse through a gelatinous
material under the influence of
an electric field.
• Since DNA is negatively
charged (phosphate groups), it
moves toward the positive
electrode.
• Shorter fragments move further
than longer ones.
Figure 20.x1a Laboratory worker reviewing DNA band pattern
DNA TECHNOLOGY TECHNIQUES USED TO
ANALYZE DNA sequences
DNA Fingerprint
• Pattern of bands, arranged in colums, made
up of specific fragments from an individual’s
DNA.
• Can be used to:
1. compare samples of blood or tissue left at a
crime scene;
2. determine how closely related species are;
3. Paternity testing.
Making a DNA Fingerprint
RFLP analysis
•
•
•
•
A DNA sample is extracted from nucleated cells.
The DNA is amplified using P.C.R.
The DNA is cut into fragments by restriction enzymes.
The stained fragments are placed into a gel, and are
moved by an electrical current.
• Smaller fragments migrate the furthest and the result is a
column of dark DNA bands that extend across the gel.
• The amount of DNA between restriction sites varies from
individual to individual of the same species. The
differences are called restriction fragment length
polymorphisms or RFLP’s. RFLP’s result in unique
restriction fragment patterns on a gel.
APPLICATIONS
Gel Electrophoresis
1. Compare DNA fragments of closely related species
to determine evolutionary relationships.
2. CSI. Compare restriction fragments between
individuals of the same species!
Fragments differ in length because of polymorphisms, slight
differences in DNA sequences. These fragments are called
restriction fragment length polymorphisms, or RFLP’s.
Figure 20.17 DNA fingerprints from a murder case