Download ch. 12 Biotechnology-notes-ppt

Biotechnology
Pre-AP Biology
Ch.12
Ms. Haut
DNA technology has many useful
applications
– The Human Genome Project
– The production of vaccines, cancer drugs, and
pesticides
– Engineered
bacteria that
can clean up
toxic wastes
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
•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)
BACTERIAL PLASMIDS AND
GENE CLONING
•Plasmids are used to customize bacteria: An
overview
– Gene cloning is one application of DNA
technology
•
Methods for studying and manipulating
genetic material
The Bacterial Chromosome
• One double-stranded,
circular molecule of
DNA
• Located in nucleoid
region, so
transcription and
translation can
occur simultaneously
• Many also contain
extrachromosomal DNA
in plasmids
Binary Fission
Plasmids
• Short, circular DNA molecules outside the
chromosome
• Carry genes that are beneficial but not
essential
• Replicate independently of chromosome
R Plasmids
• Contain genes that confer antibiotic
resistance
• Medical consequences:resistant strains of
pathogens due to overuse of antibiotics
Genetic Recombination Produces
New Bacterial Strain
• Transformation
• Transduction
• Conjugation
Gene transfer
occurs separately
from bacterial
reproduction
Bacteria as Tools
• Bacterial Transformation—
– Uptake of DNA from the fluid surrounding the
cell
– Causes genetic
recombination
Transformation
• Alteration of bacterial cell’s genotype by
uptake of naked, foreign DNA from the
environment
Transformation
• Biotech companies use this technique to
artificially introduce foreign genes into
bacterial genomes (human insulin, human
growth hormone)
Transduction
• Gene transfer from
one bacterium to
another by a
bacteriophage
Conjugation
• Direct transfer of genetic material between
bacterial cells that are temporarily joined
(bacterial sex)
“male”
“female”
F-
F+
Sex pili
“Maleness” results from
presence of F factor—
segment of DNA in
chromosome or in F plasmid
Mating bridge
Sex pili
Donor cell
(“male”)
Figure 12.1C
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
Recipient cell
(“female”)
• The transferred DNA is then integrated into the
recipient cell’s chromosome
Donated DNA
Degraded DNA
Crossovers
Recipient cell’s
chromosome
Figure 12.1D
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
Recombinant
chromosome
Bacterial plasmids can serve as
carriers for gene transfer
F factor (integrated)
Male (donor) cell
Origin of F replication
Bacterial chromosome
• An F factor is a DNA
segment in bacteria that
enables conjugation and
contains an origin of
replication
Copyright © 2003 Pearson Education, Inc. publishing Benjamin Cummings
Figure 12.2A
F factor starts
replication and
transfer of chromosome
Recipient cell
Only part of the
chromosome transfers
Recombination can occur
– Researchers can insert desired genes into
plasmids, creating recombinant DNA
•
Figure 12.1
And insert those plasmids into bacteria
(transformation)
E. coli
– If the recombinant
bacteria multiply
into a clone
• The foreign
genes are
also copied
1 Isolate DNA
Human cell
from two sources
2 Cut both
Plasmid
DNAs with
the same
restriction
enzyme
DNA
Gene V
Sticky ends
3 Mix the DNAs; they join
by base-pairing
4 Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
5 Put plasmid into bacterium
by transformation
6 Clone the bacterium
Bacterial clone carrying many
copies of the human gene
•Restriction Enzymes are used to “cut and paste”
DNA
– The tools used to make recombinant DNA are
•
•
Restriction enzymes, which cut DNA at specific
sequences
DNA ligase, which “pastes” DNA fragments together
– Creating
recombinant DNA
using restriction
enzymes and DNA
ligase
Figure 12.2
Genes can be cloned in recombinant
plasmids: A closer look
– Bacteria take the
recombinant
plasmids from their
surroundings
– And reproduce,
thereby cloning the
plasmids and the
genes they carry
•Cloned genes can be stored in genomic libraries
– Genomic libraries, sets of DNA fragments
containing all of an organism’s genes
•
Can be constructed and stored in cloned bacterial
plasmids or phages
Genome cut up with
restriction enzyme
Recombinant
plasmid
Recombinant
phage DNA
or
Bacterial
clone
Figure 12.4
Plasmid library
Phage
clone
Phage library
CONNECTION
•Recombinant cells and
organisms can mass-produce
gene products
– Applications of gene
cloning include
•
The mass
production of gene
products for
medical and other
uses
Table 12.6
Genetically modified organisms are
transforming agriculture
• New genetic varieties of animals and plants are being
produced
– A plant with a new trait can be created using the Ti
plasmid
• Biotech companies can artificially induce
transformation of bacteria
• “Golden rice” has been genetically modified to
contain beta-carotene
– This rice could help prevent vitamin A deficiency
Figure 12.18B
Drought resistant corn
Flavr Savr Tomato (CalGene)
•Transgenic organisms
– Are those that have had genes from other
organisms inserted into their genomes
– Different organisms, including bacteria, yeast,
and mammals
•
Can be used for this purpose
These sheep carry a
gene for a human
blood protein that is a
potential treatment for
cystic fibrosis
Figure 12.6
CONNECTION
•DNA technology is changing the
pharmaceutical industry
– DNA technology
•
Is widely used to produce medicines and to
diagnose diseases
DNA technology is changing the
pharmaceutical industry and medicine
• Hormones, cancer-fighting drugs, and new
vaccines are being produced using DNA
technology
– This lab equipment
is used to produce
a vaccine against
hepatitis B
Figure 12.17
•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
•Diagnosis and Treatment of Disease
– DNA technology
•
Is being used increasingly in disease diagnosis
•Vaccines
– DNA technology
•
Is also helping medical researchers develop
vaccines
Figure 12.7B
Gene therapy may someday help
treat a variety of diseases
• Techniques for manipulating
DNA have potential for treating
disease by altering an afflicted
individual’s genes
• 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
– Progress is slow, however
– There are also ethical questions
related to gene therapy
3 Viral DNA
inserts into
chromosome
Bone marrow
cell from patient
Bone
marrow
4 Inject cells
Figure 12.19
into patient
DNA Fingerprinting
• A method of
developing a person’s
DNA “profile,” similar
to a fingerprint.
• Pioneered in England
in 1984 by Dr. Alec
Jeffreys
Dr. Alec Jeffreys
First Forensic Use
• First used by law
enforcement in England in
the mid-1980’s.
• DNA evidence exonerated
one man, and convicted
another.
• Described in The Blooding,
by Joseph Wambaugh
How does it work?
• 99.9% of your DNA is the same as everyone
else’s.
• The 0.1% that differs are a combination of:
– Gene differences (Differences in the genes
themselves)
– Differences in “polymorphic regions” between
the genes on the DNA.
How does it work?
• Certain points between the genes on the
DNA have repeating base sequences.
– For example:
ATTACGCGCGCGCGCGCGCTAGC
– These are called variable nucleotide tandem
repeats (VNTRs for short)
How does it work?
• Everyone has VNTRs at the same place in
their DNA, but they are different lengths for
different people.
– For example:
Person 1: ATTACGCGCGCGCGCGCGTAGC
(7 repeats)
Person 2: ATTACGCGCGCGCGTAGC
(5 repeats)
To Make a DNA Fingerprint…
• First, we use restriction enzymes to chop the
DNA up into millions of fragments of
various lengths.
– The restriction enzymes cut everyone’s DNA in
the same place.
– Some of the fragments contain VNTRs; some
do not. The ones that do are different lengths
for different people.
Restriction Fragment Length
Polymorphisms (RFLPs)
• Polymorphisms are slight differences in
DNA sequences as seen in individuals of the
same species
To Make a DNA Fingerprint…
• Next, we use gel
electrophoresis to
sort the DNA
fragments by size.
Gel Electrophoresis
• Method for sorting proteins or nucleic acids
on the basis of their electric charge and size
Gel Electrophoresis
Electrical current carries
negatively-charged DNA
through gel towards
positive electrode
• Agarose gel sieves DNA
fragments according to
size
•
–
Small fragments move
farther than large fragments
Gel Electrophoresis
1
To Make a DNA
Fingerprint…
Restriction fragment
preparation
Restriction
fragments
2
Gel electrophoresis
3
Blotting
4
Radioactive probe
Filter paper
Probe
5
Detection of radioactivity
(autoradiography)
Film
Figure 12.11C
• Finally, a radioactive
probe attaches to our
VNTRs. Only the
fragments with our
VNTRs will show up
on the gel.
To Make a DNA Fingerprint…
• Since VNTRS are
different lengths in
different people, this
creates a DNA
Fingerprint.
Two uses for DNA Fingerprints...
• Forensics
DNA taken from crime
scenes (blood, semen, hair,
etc.) can be compared to the
DNA of suspects.
Real-life CSI!
Two uses for DNA Fingerprints...
• Forensics
This is an example of a gel
that might be used to
convict a rape suspect.
Compare the “Sperm DNA”
to the “Suspect DNA.”
Which suspect committed
the rape?
Two uses for DNA Fingerprints...
• Paternity Testing
Since all of our DNA
markers came from
either mommy or
daddy, we can use
DNA fingerprints to
determine whether a
child and alleged
father are related…just
like on Maury Povich!
Interpreting DNA Fingerprints
• A blood stain was found at
a murder scene. The blood
belongs to which of the
seven possible suspects?
Interpreting DNA Fingerprints
• Who committed this
rape?
Interpreting DNA Fingerprints
• These DNA fingerprints
are from a mother, a child,
and two possible
biological fathers. Which
one is the daddy?
Interpreting DNA Fingerprints
• Mother, father, and
four children. Which
child is from a
different father?
DNA Fingerprinting
– A nucleic acid probe
•
•
Is a short, single-stranded molecule of
radioactively labeled or fluorescently labeled
DNA or RNA
Can tag a desired gene in a library
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Figure 12.8
Base pairing
indicates the
gene of interest
RESTRICTION FRAGMENT ANALYSIS
AND DNA FINGERPRINTING
•Nucleic acid probes identify clones carrying
specific genes
– DNA technology methods
•
Can be used to identify specific pieces of DNA
CONNECTION
•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
1 mRNA
isolated
Reverse transcriptase
and fluorescent DNA
nucleotides
2 cDNA made
from mRNA
Actual size
(6,400 genes)
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
Using DNA Probes to Detect Harmful
Alleles
1 Restriction fragment
preparation
I
•Radioactive probes
II
III
Restriction
fragments
– Can reveal DNA bands
of interest on a gel
•Detecting a harmful
allele using restriction
fragment analysis
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
Polymerase Chain Reaction
•The PCR method is
used to amplify DNA
sequences
– Can be used to clone a
small sample of DNA
quickly, producing
enough copies for
analysis
– Does not rely on cells
for DNA replication
Figure 12.14
PCR Reaction
GENOMICS CONNECTION
•The Human Genome Project is an ambitious
application of DNA technology
– The Human Genome Project, begun in 1990 and
now largely completed, involved
•
Genetic and physical mapping of chromosomes,
followed by DNA sequencing
Figure 12.15
CONNECTION
•The science of genomics compares whole
genomes
– The sequencing of many prokaryotic and
eukaryotic genomes
•
Has produced data for genomics, the study of
whole genomes
– Besides being interesting themselves
•
Table 12.17
Nonhuman genomes can be compared with the
human genome
Proteomics
•Is the study of the full
sets of proteins produced
by organisms
Biotechnology Explorer™
Protein Fingerprinting
Instruction Manual
Biotechnology
Explorer™
Protein
Fingerprinting
Instruction
Manual
Could GM organisms harm human
health or the environment?
• Genetic engineering involves
some risks
– Possible ecological damage from
pollen transfer between GM and
wild crops
– Pollen from a transgenic variety of
corn that contains a pesticide may
stunt or kill monarch caterpillars
Figure 12.20A, B
DNA technology raises important
ethical questions
• Our new genetic knowledge
will affect our lives in
many ways
• The deciphering of the
human genome, in
particular, raises
profound ethical issues
– Many scientists have
counseled that we
must use the
information wisely
Figure 12.21A-C
Analysis of Stained Gel
•
•
Determine restriction fragment sizes
–
Create standard curve using DNA marker
–
Measure distance traveled by restriction fragments
–
Determine size of DNA fragments
Identify the related samples
Molecular Weight Determination
Fingerprinting Standard Curve: Semi-log
Distance (mm)
23,000
11.0
9,400
13.0
6,500
15.0
4,400
18.0
2,300
23.0
2,000
24.0
100,000
10,000
Size, base pairs
Size (bp)
B
1,000
100
0
5
10
15
Distance, mm
20
A
25
30
Acknowledgements
• BIOLOGY: CONCEPTS AND CONNECTIONS 4th Edition, by
Campbell, Reece, Mitchell, and Taylor, ©2003. These images have
been produced from the originals by permission of the publisher. These
illustrations may not be reproduced in any format for any purpose
without express written permission from the publisher.
• Unless otherwise noted, illustrations are credited to Pearson Education
which have been borrowed from BIOLOGY: CONCEPTS AND
CONNECTIONS 4th Edition, by Campbell, Reece, Mitchell, and
Taylor, ©2001. These images have been produced from the originals
by permission of the publisher. These illustrations may not be
reproduced in any format for any purpose without express written
permission from the publisher.