Biotechnology

jamaicanabsorbingBiotechnology

Dec 5, 2012 (4 years and 9 months ago)

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Biotechnology

AP Biology

Ch. 20

Ms. Haut

Biotechnology


Isolation, analysis, and manipulation of DNA
has brought about many breakthroughs


Pharmaceuticals

production of insulin,
medications


Cloning

animals, organ replacements


Forensics

DNA fingerprinting


Human Genome Project


Gene Therapy


http://img.dailymail.co.uk/i/pix/2007/09_03/dnaDM1909_468x570.jpg

Bacteria as Tools


Bacteria


Circular DNA


Plasmid


Extra genetic material


Small, circular DNA


Not necessary, but
usually beneficial

http://users.rcn.com/jkimball.ma.ultranet/Biol
ogyPages/R/RecombinantPlasmid.gif

Bacteria as Tools


Bacterial
Transformation


Uptake of DNA from the
fluid surrounding the cell


Causes genetic
recombination


Allow insertion of gene
of interest

http://biology200.gsu.edu/houghton/4564%20'04/fig
ures/lecture%203/transformation.jpg

Biotech companies can
artificially induce bacterial
transformation

http://gecorn.tripod.com/sitebuildercontent/sitebuilderpictures/tr
ansgeniccrop.gif

Transgenic Plants

1994.
Flavr Savr Tomato
. 1
st

engineered food in stores.
Engineered to remain firm even
as it turns red and ripe.

Bt transgenic corn

is normal
corn that contains a gene from the
soil bacterium
Bacillus
thuringiensis
. Gene allows
production of a toxic protein that
can kill many types of caterpillars

(http://www.ces.ncsu.edu/plymouth/pubs/btcorn99.html)


How Do We Isolate DNA of
Interest?


DNA is a long molecule


To study genes of interest must isolate
fragments of DNA


Restriction Enzymes

cut DNA at specific
sequences


Using Restriction Enzymes to
Make Recombinant DNA


Bacterial restriction enzymes cut DNA
molecules at DNA sequences called
restriction sites


A restriction enzyme usually makes many
cuts, yielding
restriction fragments


The most useful restriction enzymes cut
DNA in a staggered way, producing
fragments with
“sticky ends”

that bond with
complementary “sticky ends” of other
fragments


DNA ligase is an enzyme that seals the
bonds between restriction fragments

Cloning a Eukaryotic Gene in
a Bacterial Plasmid


In gene cloning, the original plasmid is called
a
cloning vector


A cloning vector is a DNA molecule that can
carry foreign DNA into a cell and replicate
there

Producing Clones of Cells

Isolate plasmid DNA
and human DNA.
Cut both DNA samples with
the same restriction enzyme.
Mix the
DNAs
; they join by base pairing.
The products are recombinant plasmids
and many
nonrecombinant
plasmids.
Bacterial cell
lac
Z
gene
(lactose
breakdown)
Human
cell
Restriction
site
amp
R
gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own
lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X
-
gal.
Incubate until colonies grow.
Colony carrying non
-
recombinant plasmid
with intact
lacZ
gene
Colony carrying
recombinant
plasmid with
disrupted
lacZ
gene
Bacterial
clone
Isolate plasmid DNA
and human DNA.
Cut both DNA samples with
the same restriction enzyme.
Mix the
DNAs
; they join by base pairing.
The products are recombinant plasmids
and many
nonrecombinant
plasmids.
Bacterial cell
lac
Z
gene
(lactose
breakdown)
Human
cell
Restriction
site
amp
R
gene
(ampicillin
resistance)
Bacterial
plasmid
Gene of
interest
Sticky
ends
Human DNA
fragments
Recombinant DNA plasmids
Introduce the DNA into bacterial cells
that have a mutation in their own
lacZ
gene.
Recombinant
bacteria
Plate the bacteria on agar
containing ampicillin and X
-
gal.
Incubate until colonies grow.
Colony carrying non
-
recombinant plasmid
with intact
lacZ
gene
Colony carrying
recombinant
plasmid with
disrupted
lacZ
gene
Bacterial
clone
Identifying Clones Carrying a
Gene of Interest


A clone carrying the gene of interest can be
identified with a
nucleic acid probe

having a
sequence complementary to the gene


This process is called
nucleic acid
hybridization


An essential step in this process is
denaturation of the cells’ DNA, separation of
its two strands

Nucleic Acid Probe
Hybridization

Master plate
Filter
Solution
containing
probe
Filter lifted
and flipped over
Radioactive
single
-
stranded
DNA
Probe
DNA
Gene of
interest
Single
-
stranded
DNA from cell
Film
Hybridization
on filter
Master plate
Colonies
containing
gene of
interest
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to break
open the cells and denature
their DNA; the resulting
single
-
stranded DNA
molecules are treated so that
they stick to the filter.
The filter is laid
under photographic
film, allowing any
radioactive areas to
expose the film
(autoradiography).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Master plate
Filter
Solution
containing
probe
Filter lifted
and flipped over
Radioactive
single
-
stranded
DNA
Probe
DNA
Gene of
interest
Single
-
stranded
DNA from cell
Film
Hybridization
on filter
Master plate
Colonies
containing
gene of
interest
A special filter paper
is pressed against
the master plate,
transferring cells to
the bottom side of
the filter.
The filter is treated to break
open the cells and denature
their DNA; the resulting
single
-
stranded DNA
molecules are treated so that
they stick to the filter.
The filter is laid
under photographic
film, allowing any
radioactive areas to
expose the film
(autoradiography).
After the
developed film is
flipped over, the
reference marks
on the film and
master plate are
aligned to locate
colonies carrying
the gene of
interest.
Storing Cloned Genes in DNA
Libraries


A genomic library that is made using bacteria
is the collection of recombinant vector clones
produced by cloning DNA fragments from an
entire genome


A genomic library that is made using
bacteriophages is stored as a collection of
phage clones

http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/10_23_ge
nomic_library.jpg

Genomic Libraries

http://homepages.strath.ac.uk/~dfs99109/BB211/Lo
d7
-
12genomiclibrar.JPG

Plasmid Library

Phage Library

Cloning and Expressing
Eukaryotic Genes


As an alternative to screening a DNA library,
clones can sometimes be screened for a
desired gene based on detection of its
encoded protein


After a gene has been cloned, its protein
product can be produced in larger amounts
for research

Bacterial Expression Systems


Several technical difficulties hinder
expression of cloned eukaryotic genes in
bacterial host cells


To overcome differences in promoters and
other DNA control sequences, scientists
usually employ an expression vector, a
cloning vector that contains a highly active
prokaryotic promoter

Eukaryotic Cloning and
Expression Systems


The use of cultured eukaryotic cells as host
cells and yeast artificial chromosomes
(YACs) as vectors helps avoid gene
expression problems


YACs behave normally in mitosis and can
carry more DNA than a plasmid


Eukaryotic hosts can provide the
posttranslational modifications that many
proteins require


Introducing Recombinant DNA
into Eukaryotic Cells


Electroporation

brief
electrical pulse creates
temporary holes in
plasma membranes


Alternatively, scientists
can inject DNA into
cells using microscopic
needles


Once inside the cell,
the DNA is
incorporated into the
cell’s DNA by natural
genetic recombination

http://www.cyagra.com/process3.htm

http://www.nature.com/ki/journal/v61/n1s/images/4493051f1b.gif

Amplifying DNA
in Vitro
: The
Polymerase Chain Reaction
(PCR)


The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA


A three
-
step cycle

heating, cooling, and
replication

brings about a chain reaction
that produces an exponentially growing
population of identical DNA molecules

Polymerase Chain
Reaction (PCR)


Quick method of
cloning DNA in
vitro (without
using cells)


Can be used to
increase small
amounts of DNA
for analysis

Genomic DNA
Target
sequence
5

3

3

5

5

3

3

5

Primers
Denaturation
:
Heat briefly
to separate DNA
strands
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3

end of each
primer
Cycle 1
yields
2
molecules
New
nucleo
-
tides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
Genomic DNA
Target
sequence
5

3

3

5

5

3

3

5

Primers
Denaturation
:
Heat briefly
to separate DNA
strands
Annealing:
Cool to allow
primers to form
hydrogen bonds
with ends of
target sequence
Extension:
DNA polymerase
adds nucleotides to
the 3

end of each
primer
Cycle 1
yields
2
molecules
New
nucleo
-
tides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
PCR Reaction

Restriction fragment analysis detects
DNA differences that affect
restriction sites


Restriction fragment
analysis

detects
differences in the
nucleotide sequences of
DNA molecules


Such analysis can rapidly
provide comparative
information about DNA
sequences

DNA + restriction enzyme
Restriction
fragments

Normal

-
globin
allele


Sickle
-
cell
allele


Heterozygote
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.





Nitrocellulose
paper (blot)
Gel
Sponge
Alkaline
solution
Paper
towels
Heavy
weight
Hybridization with radioactive probe.





Radioactively
labeled probe
for

-
globin
gene is added
to solution in
a plastic bag
Paper blot
Probe hydrogen
-
bonds to fragments
containing normal
or mutant

-
globin
Fragment from
sickle
-
cell

-
globin
allele
Fragment from
normal

-
globin
allele
Autoradiography.





Film over
paper blot
DNA + restriction enzyme
Restriction
fragments

Normal

-
globin
allele


Sickle
-
cell
allele


Heterozygote
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.





Nitrocellulose
paper (blot)
Gel
Sponge
Alkaline
solution
Paper
towels
Heavy
weight
Hybridization with radioactive probe.





Radioactively
labeled probe
for

-
globin
gene is added
to solution in
a plastic bag
Paper blot
Probe hydrogen
-
bonds to fragments
containing normal
or mutant

-
globin
Fragment from
sickle
-
cell

-
globin
allele
Fragment from
normal

-
globin
allele
Autoradiography.





Film over
paper blot
Gel Electrophoresis


One indirect method of
rapidly analyzing and
comparing genomes is
gel electrophoresis


This technique uses a
gel as a molecular
sieve to separate
nuclei acids or proteins
by size

http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.gif

Agarose Gel Electrophoresis


Electrical current
carries
negatively
-
charged
DNA through
gel towards positive
(red) electrode


Agarose gel sieves
DNA fragments
according to size


Small fragments move
farther than large
fragments

Restriction Fragment Analysis


DNA fragments
produced by restriction
enzyme digestion of a
DNA molecule are
sorted by gel
electrophoresis


Restriction fragment
analysis is useful for
comparing two different
DNA molecules, such
as two alleles for a
gene

Normal

-
globin
allele
175
bp
201
bp
Large fragment
Sickle
-
cell mutant

-
globin
allele
376
bp
Large fragment
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
restriction sites in normal and sickle
-
cell alleles of

-
globin
gene
Normal
allele
Sickle
-
cell
allele
Large
fragment
376
bp
201
bp
175
bp
Electrophoresis of restriction fragments from normal
and sickle
-
cell alleles
Southern Blotting


A technique called
Southern blotting

combines gel
electrophoresis with
nucleic acid
hybridization


Specific DNA
fragments can be
identified by
Southern blotting,
using labeled probes
that hybridize to the
DNA immobilized on
a “blot” of gel

DNA + restriction enzyme
Restriction
fragments

Normal

-
globin
allele


Sickle
-
cell
allele


Heterozygote
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.





Nitrocellulose
paper (blot)
Gel
Sponge
Alkaline
solution
Paper
towels
Heavy
weight
Hybridization with radioactive probe.





Radioactively
labeled probe
for

-
globin
gene is added
to solution in
a plastic bag
Paper blot
Probe hydrogen
-
bonds to fragments
containing normal
or mutant

-
globin
Fragment from
sickle
-
cell

-
globin
allele
Fragment from
normal

-
globin
allele
Autoradiography.





Film over
paper blot
DNA + restriction enzyme
Restriction
fragments

Normal

-
globin
allele


Sickle
-
cell
allele


Heterozygote
Preparation of restriction fragments.
Gel electrophoresis.
Blotting.





Nitrocellulose
paper (blot)
Gel
Sponge
Alkaline
solution
Paper
towels
Heavy
weight
Hybridization with radioactive probe.





Radioactively
labeled probe
for

-
globin
gene is added
to solution in
a plastic bag
Paper blot
Probe hydrogen
-
bonds to fragments
containing normal
or mutant

-
globin
Fragment from
sickle
-
cell

-
globin
allele
Fragment from
normal

-
globin
allele
Autoradiography.





Film over
paper blot
Restriction Fragment Length
Differences as Genetic
Markers


Restriction fragment length polymorphisms

(
RFLPs
, or Rif
-
lips) are differences in DNA
sequences on homologous chromosomes
that result in restriction fragments of
different lengths


A RFLP can serve as a genetic marker for a
particular location (locus) in the genome


RFLPs are detected by Southern blotting

Restriction Fragment Analysis


Can be used to determine
paternity.


Cutting an individual’s
DNA with a restriction
enzyme creates a “DNA
fingerprint”

Restriction Fragment Analysis


Can be used as evidence
in criminal cases


Can compare DNA
samples from the
defendant, the victim, and
the defendant’s clothing


Requires only tiny
amounts of blood or other
tissue

Entire genomes can be
mapped at the DNA level


The most ambitious mapping project to date
has been the sequencing of the human
genome


Officially begun as the Human Genome
Project in 1990, the sequencing was largely
completed by 2003


Scientists have also sequenced genomes of
other organisms, providing insights of
general biological significance

Genetic (Linkage) Mapping:
Relative Ordering of Markers


The first stage in
mapping a large
genome is constructing
a linkage map of
several thousand
genetic markers
throughout each
chromosome


The order of markers
and relative distances
between them are
based on
recombination
frequencies

Cytogenetic
map
Genes located
by FISH
Chromosome
bands
Genetic
markers
Genetic (linkage)
mapping
Physical mapping
Overlapping
fragments
DNA sequencing
Physical Mapping: Ordering
DNA Fragments


A physical map is constructed by cutting a
DNA molecule into many short fragments
and arranging them in order by identifying
overlaps


Physical mapping gives the actual distance
in base pairs between markers

Genetic Maps


http://www.informatics.jax.org/silver/images/figure7
-
1.gif

DNA Sequencing


Relatively short DNA fragments can be
sequenced by the dideoxy chain
-
termination
method


Inclusion of special dideoxyribonucleotides in
the reaction mix ensures that fragments of
various lengths will be synthesized

DNA Sequencing

DNA
(template strand)
5

3

Primer
3

5

DNA
polymerase
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
3

5

DNA (template
strand)
Labeled strands
3

Direction
of movement
of strands
Laser
Detector
DNA Sequencing

http://files.myweb.med.ucalgary.ca/files/64/images/DNA%20Sequencing%20Images/Sam
ple_sequencing_result_2005
-
10
-
25_copy.jpg


Linkage mapping, physical mapping, and
DNA sequencing represent the overarching
strategy of the Human Genome Project


An alternative approach to sequencing
genomes starts with sequencing random
DNA fragments


Computer programs then assemble
overlapping short sequences into one
continuous sequence

Whole
-
Genome Shotgun
Approach to Sequencing

Cut the DNA from
many copies of an
entire chromosome
into overlapping
frag
-
ments
short enough
for sequencing
Clone the fragments
in plasmid or phage
vectors
Sequence each fragment
Order the
sequences into one
overall sequence
with computer
software
Genome sequences provide
clues to important biological
questions


In genomics, scientists study whole sets of
genes and their interactions


Genomics is yielding new insights into
genome organization, regulation of gene
expression, growth and development, and
evolution

Identifying Protein
-
Coding
Genes in DNA Sequences


Computer analysis of genome sequences
helps identify sequences likely to encode
proteins


The human genome contains about 25,000
genes, but the number of human proteins is
much larger


Comparison of sequences of “new” genes
with those of known genes in other species
may help identify new genes

Determining Gene Function


One way to determine function is to disable
the gene and observe the consequences
(knock
-
outs)


Using
in vitro

mutagenesis, mutations are
introduced into a cloned gene, altering or
destroying its function


When the mutated gene is returned to the
cell, the normal gene’s function might be
determined by examining the mutant’s
phenotype

A transgenic mouse with an active rat
growth hormone gene (
left
). This
transgenic mouse is twice the size of a
normal mouse (
right
).

http://web.virginia.edu/Heidi/chapter29/Images/8883n29_30.jpg


Studying Expression of
Interacting Groups of Genes


Automation has
allowed scientists to
measure expression of
thousands of genes at
one time using
DNA
microarray assays


DNA microarray assays
compare patterns of
gene expression in
different tissues, at
different times, or
under different
conditions

Make
cDNA
by reverse
transcription, using
fluorescently labeled
nucleotides.
Apply the
cDNA
mixture to a
microarray
, a microscope slide
on which copies of single
-
stranded DNA fragments from
the organism

s genes are fixed,
a different gene in each spot.
The
cDNA
hybridizes with any
complementary DNA on the
microarray
.
Rinse off excess
cDNA
; scan
microarray
for fluorescent.
Each fluorescent spot
(yellow) represents a gene
expressed in the tissue
sample.
Isolate mRNA.
Tissue sample
mRNA molecules
Labeled
cDNA
molecules
(single strands)
DNA
microarray
Size of an actual
DNA
microarray
with all the genes
of yeast (6,400 spots)
DNA Microarray Analysis

Comparing Genomes of
Different Species


The more similar the nucleotide sequences
between two species, the more closely
related these species are in their
evolutionary history


Comparative genome studies confirm the
relevance of research on simpler organisms
to understanding human biology

Future Directions in
Genomics


Genomics is the study of entire genomes


Proteomics is the systematic study of all
proteins encoded by a genome


Single nucleotide polymorphisms (SNPs)
provide markers for studying human genetic
variation

The practical applications of
DNA technology affect our
lives in many ways


Many fields benefit from DNA technology and
genetic engineering

Medical Applications


One benefit of DNA technology is
identification of human genes in which
mutation plays a role in genetic diseases

Diagnosis of Diseases

Normal

-
globin
allele
175
bp
201
bp
Large fragment
Sickle
-
cell mutant

-
globin
allele
376
bp
Large fragment
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
Dd
el
restriction sites in normal and sickle
-
cell alleles of

-
globin
gene
Normal
allele
Sickle
-
cell
allele
Large
fragment
376
bp
201
bp
175
bp
Electrophoresis of restriction fragments from normal
and sickle
-
cell alleles

Scientists can diagnose
many human genetic
disorders by using PCR and
primers corresponding to
cloned disease genes, then
sequencing the amplified
product to look for the
disease
-
causing mutation


Even when a disease gene
has not been cloned,
presence of an abnormal
allele can be diagnosed if a
closely linked RFLP marker
has been found

Human Gene Therapy


Alteration of an afflicted
individual’s genes


Holds great potential
for treating disorders
traceable to a single
defective gene


Vectors are used for
delivery of genes into
cells


Raises ethical
questions, such as
whether human germ
-
line cells should be
treated to correct the
defect in future
generations

Cloned gene
Retrovirus
capsid
Bone
marrow
cell from
patient
Inject engineered
cells into patient.
Insert RNA version of normal allele
into retrovirus.
Viral RNA
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
Pharmaceutical Products


Some pharmaceutical
applications of DNA
technology:


Large
-
scale production
of human hormones and
other proteins with
therapeutic uses


Production of safer
vaccines

Forensic Evidence


DNA “fingerprints” obtained by analysis of
tissue or body fluids can provide evidence in
criminal and paternity cases


A DNA fingerprint is a specific pattern of
bands of RFLP markers on a gel


The probability that two people who are not
identical twins have the same DNA
fingerprint is very small


Exact probability depends on the number of
markers and their frequency in the
population

Environmental Cleanup


Genetic engineering can be used to modify
the metabolism of microorganisms


Some modified microorganisms can be used
to extract minerals from the environment or
degrade potentially toxic waste materials

Animal Husbandry


Transgenic organisms

are
made by introducing
genes from one species
into the genome of
another organism


May be created to exploit
the attributes of new genes
(such as genes for faster
growth or larger muscles)


Other transgenic organisms
are pharmaceutical
“factories,” producers of
large amounts of otherwise
rare substances for medical
use

Genetic Engineering in Plants


Agricultural scientists
have endowed a
number of crop plants
with genes for
desirable traits


The Ti plasmid is the
most commonly used
vector for introducing
new genes into plant
cells

Agrobacterium
tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
DNA with
the gene
of interest
T DNA
Recombinant
Ti plasmid
Plant with
new trait
Safety and Ethical Questions
Raised by DNA Technology


Potential benefits of genetic engineering
must be weighed against potential hazards of
creating harmful products or procedures


Most public concern about possible hazards
centers on genetically modified (GM)
organisms used as food