+
DNA
Technology and Genetic
Engineering
Human protein
production
RFLP analysis
Human genome
project
Transgenic
organisms
Forensic analysis
+
DNA Technology and Genetic
Engineering (making changes in DNA)
1: Production of human proteins
2: To identify people
3: To identify human diseases
4: To identify all human genes
5: To genetically engineer food
Supercoils
Coils
Nucleosomes
Histones
Nucleus
Chromosome
DNA
Cell
+
Use 1: To produce human
proteins in bacteria (
E. coli
)
Example: Curing Pituitary Dwarfism
+
What is Dwarfism?
Dwarfism is a recessive
disease that causes adults to
be no more than 4 feet tall
Little people produce little
or no growth hormone,
which is made by the
pituitary
Researchers studied families
with dwarfism and found that
people with dwarfism have
defective copies of the gene,
GH1
+
Early attempts to treat Dwarfism
Attempts to inject growth hormone from pigs (a
strategy that worked previously for insulin) did not
work
–
only GH from humans would work (until
1982 source from human cadavers and up to 20,000
pituitaries were needed!)
Some of these pituitaries were contaminated with
prions, which cause degenerative brain disorders
+
Solution: Use recombinant DNA to
produce GH in bacteria!
Recombinant DNA = DNA that
results from combining DNA from
different sources
ex. mouse + human DNA
human + bacterial DNA
+
Plasmid
isolated
1
Bacterium
Bacterial
chromosome
Plasmid
2
DNA
isolated
Cell containing gene
of interest
DNA
Gene of
interest
3
Gene
inserted
into plasmid
Recombinant DNA
(plasmid)
4
Plasmid put into
bacterial cell
Recombinant
bacterium
5
Copies of gene
Copies of protein
Clones of cell
Gene for pest
resistance
inserted into
plants
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to dissolve blood
clots in heart attack therapy
Protein used to
make snow form
at higher
temperature
Cell multiplies with
gene of interest
Recombinant DNA Overview
+
How Do You Make Recombinant
DNA?
How do you make recombinant DNA? We need
A) To isolate genes with restriction enzymes
B) A vector
C) To combine the genes and the vector
What do we do with it?
D) Transfer the recombinant DNA to the host
E) Find the gene of interest (human growth
hormone)
+
A) Isolate genes with restriction
enzymes (DNA scissors)
Occur naturally in
bacteria
–
why?
Bacteriophages
infect
bacteria
Cut up foreign DNA
Hundreds are purified
and available
commercially
Recognize and cut at
specific base sequences
in DNA (usually 4
-
8
bases long)
+
Products generated by restriction
enzymes
A) Sticky
-
end cutters
Enzyme
Recognition site
DNA after cuts
B) Blunt
-
end cutters
Enzyme
Recognition site
DNA after cuts
5’...G
3’...CTTAA
AATTC
...3’
G
...5’
5’...CCC
3’...GGG
GGG
...3’
CCC
...5’
5’...CCC
GGG
...3’
3’...GGG
CCC
...5’
SmaI
EcoRI
5’...G
AATTC
...3’
3’...CTTAA
G
...5’
+
A) Isolate genes with restriction
enzymes (DNA scissors)
Take genomic DNA (in this case, human cells) and cut
it with a particular restriction enzyme
MANY restriction fragments formed
ALL parts of the DNA are cut whenever there is a
restriction site
Now what? Put these fragments in a vector
+
B) Vectors
Vector = something to carry the gene of
interest into the host (i.e. bacteria)
A. Mechanical
–
micropipettes or gene
guns
B. Biological
–
virus or plasmid
Plasmid
–
additional, free
-
floating
ring
of
DNA found only in bacteria
Can replicate within a cell (has origin of
replication)
Has antibiotic resistance gene
Cut both the plasmid and gene with the
same restriction enzyme and their ends will
hydrogen bond = gene splicing
+
C) Combine
the gene of
interest and
the vector by
sealing ends
with DNA
ligase
… now
you have made
recombinant
DNA
DNA
1
Restriction enzyme
recognition sequence
Restriction enzyme
cuts the DNA into
fragments
Sticky end
2
3
4
5
Restriction enzyme
cuts the DNA into
fragments
Addition of a DNA
fragment from
another source
Two (or more)
fragments stick
together by
base
-
pairing
DNA ligase
pastes the strand
Recombinant DNA molecule
+
C) Combine
the gene of
interest and
the vector
–
a
different
picture
+
D) Transfer the Recombinant DNA
to the Host (Transformation)
Recombinant DNA is transferred to a host cell.
Can use heat
-
shocking or electricity to get plasmid
into bacteria
How do we know which bacterial cells have our
plasmid?
Antibiotic resistance gene!
Resistance gene allows ONLY those bacteria with the
plasmid to grow in media that have an antibiotic on it
When the host cell copies its DNA (replicates), it also
makes a copy of the plasmid. Results in clones, which
are genetically identical copies.
+
D) Transfer the Recombinant DNA
to the Host
We use bacteria because they reproduce very
quickly and have all the protein synthesis
machinery (enzymes,
ribosomes
)
Insulin for Type I diabetics
Blood factor VIII
-
hemophilia (clotting factor)
Antigens for vaccines (
Hep
B, flu, meningitis)
Cutting chromosomes in order to study
individual pieces
Host Cells Produce Protein Products
–
ex. GH and insulin
+
E) Find the gene of interest
Each clone consists of
identical cells containing one
fragment (of many) of human
DNA
The collection of all the
cloned DNA fragments is
known as a genomic library
Each fragment represents a
“book” that is “shelved” in
plasmids inside bacterial
cells
Thus, it is a library of all the
organism’s genes
Figure 12.6
Genome cut up
with restriction
enzyme
Recombinant
plasmid
OR
Recombinant
phage DNA
Bacterial
clone
Phage
clone
Phage
library
Plasmid
library
+
E) *Side note
–
libraries can also
be made from
cDNA
The enzyme reverse transcriptase can be used to make a
smaller library, called a
cDNA
library
This contains only the genes that are expressed
(transcribed) by a specific cell type (rather than ALL the
genes found in an organism)
These genes can then be digested and placed in vectors
Why is this useful?
Bacterial mRNA does not have
introns
–
doesn’t have the
machinery to splice eukaryotic genes
Can help a researcher study the genes responsible for
specialized functions of a certain cell type
+
Transcription
1
CELL NUCLEUS
DNA of
eukaryotic
gene
RNA
transcript
mRNA
Exon
Intron
Exon
Intron
Exon
TEST TUBE
Reverse transcriptase
cDNA strand
cDNA of gene
(no introns)
RNA splicing
(removes introns)
2
Isolation of mRNA
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
3
Breakdown of RNA
4
Synthesis of second
DNA strand
5
+
E) Find the gene of interest
How do we find the right “shelf” in the library?
If we know at least part of the DNA sequence, we can
create a nucleic acid probe
Probe = radioactively labeled single
-
stranded DNA piece
that can base pair with a particular sequence
Ex: AT
CC
GA
The probe is mixed with clones that have been heated or
treated with a chemical to separate the DNA strands
The probe will “tag” the correct “shelf” or bacterial clone
that contains the gene of interest
+
E) Find the gene of interest
Radioactive
probe (DNA)
Mix with single
-
stranded DNA from
various bacterial
(or phage) clones
Single
-
stranded
DNA
Base pairing
indicates the
gene of interest
+
E) Find the
gene of
interest
Bacterial colonies containing
cloned segments of foreign DNA
Radioactive DNA
Transfer
cells to
filter
1
Solution
containing
probe
Treat cells
on filter to
separate
DNA
strands
2
Add probe
to filter
3
Probe
DNA
Gene of
interest
Single
-
stranded
DNA from cell
Hydrogen
-
bonding
Autoradiography
4
Developed film
Colonies of living
cells containing
gene of interest
Compare autoradiograph
with master plate
5
Master plate
Filter
paper
The bacterial
colony can
then be
grown and
the protein of
interest can
be isolated in
large
amounts
+
E) Isolating
the gene of
interest
Isolate DNA
from two sources
1
E. coli
Cut both
DNAs with
the same
restriction
enzyme
2
Plasmid
Human cell
DNA
Gene
V
Sticky ends
Mix the DNAs; they join
by base
-
pairing
3
Add DNA ligase
to bond the DNA covalently
4
Recombinant DNA
plasmid
Gene
V
Put plasmid into bacterium
by transformation
5
Clone the bacterium
6
Bacterial clone carrying many
copies of the human gene
+
Mass
-
Produced Genes
Enter the password to open this PDF file:
File name:
-
File size:
-
Title:
-
Author:
-
Subject:
-
Keywords:
-
Creation Date:
-
Modification Date:
-
Creator:
-
PDF Producer:
-
PDF Version:
-
Page Count:
-
Preparing document for printing…
0%
Σχόλια 0
Συνδεθείτε για να κοινοποιήσετε σχόλιο