Download Recombinant DNA

Biotechnology
Chapter 10
10.1 Impacts/Issues
Golden Rice or Frankenfood?
 Genes from one species may be inserted into an
individual of another species – or a gene may be
modified and reinserted into an individual of the
same species
Golden Rice
 Rice plants with added genes make and store
beta carotene
Video: Golden rice or Frankenfood?
GMOs and Transgenic Organisms
 Transgenic
• An organism that has been genetically modified
with genes from a different species
 Genetically modified organisms (GMOs)
• Organism whose genome has been modified by
genetic engineering
10.2 Finding Needles in Haystacks
 Gene research was limited until enzymes
produced by bacteria to cut viral DNA were
discovered
 Restriction enzyme
• Enzyme that cuts DNA at specific base sequences
• Used in DNA cloning to cut DNA into pieces that
are inserted into cloning vectors
DNA Cloning
 DNA cloning mass-produces DNA fragments for
research
 DNA cloning
• Set of procedures that uses living cells to make
many identical copies of a DNA fragment
 Clone
• A genetically identical copy of DNA, a cell, or an
organism
Cloning Vectors
 Cloning vector
• A DNA molecule that can accept foreign DNA,
resulting in a hybrid molecule that can be
transferred to a host cell, and get replicated in it
 Plasmid
• A small, circular DNA molecule in bacteria,
replicated independently of the chromosomes
• A cloning vector
Recombinant DNA
 Recombinant DNA molecules are introduced into
host cells such as bacteria, which copy the DNA
as they divide
 Recombinant DNA
• Contains genetic material from more than one
organism
Making Recombinant DNA
1. A restriction enzyme recognizes specific base
sequences in DNA from two different sources
2. Restriction enzymes cut DNA into fragments
with single-stranded tails (“sticky ends”)
3. DNA fragments from different sources are mixed
together; matching sticky ends base-pair
4. DNA ligase joins fragments, forming
recombinant DNA
Making Recombinant DNA
restriction
enzyme (cut)
1 A restriction enzyme
recognizes a specific base
sequence in DNA (green
boxes) from two sources.
mix
2 The enzyme cuts DNA
from both sources into
fragments that have
sticky ends.
DNA ligase
(paste)
3 The DNA fragments
from the two sources
are mixed together.
The matching sticky
ends base-pair with
each other.
4 DNA ligase joins
the fragments of
DNA where they
overlap. Molecules of
recombinant DNA
are the result.
Stepped Art
Fig. 10-2, p. 181
Commercial Plasmid Cloning Vector
Kpn l
Sph l
Pst l
Bam Hl
Eco RI
Sal l
pDrive Cloning Vector
3.85 kb
Acc l
Xho l
Xba l
Bst XI
Sac l
Not l
Fig. 10-3, p. 181
cDNA Cloning
 RNA cannot be cloned directly; reverse
transcriptase is used to copy single-stranded
RNA into cDNA for cloning
 Reverse transcriptase
• Viral enzyme that uses mRNA as a template to
make a strand of DNA
 cDNA
• DNA synthesized from an RNA template by the
enzyme reverse transcriptase
Making cDNA
mRNA
A The enzyme reverse
transcriptase transcribes
mRNA into DNA.
mRNA
cDNA
B DNA polymerase replicates
the DNA strand.
cDNA
cDNA
Eco RI recognition site
C The result is a double-stranded molecule
of DNA that can be cut and pasted into a
cloning vector.
Fig. 10-4, p. 182
mRNA
A The enzyme reverse
transcriptase transcribes
mRNA into DNA.
mRNA
cDNA
B DNA polymerase replicates
the DNA strand.
cDNA
cDNA
Eco RI recognition site
C The result is a double-stranded molecule
of DNA that can be cut and pasted into a
cloning vector.
Stepped Art
Fig. 10-4, p. 182
Libraries
 A library is a collection of cells that host different
fragments of DNA, often representing an
organism’s entire genome
 Researchers make DNA libraries to isolate one
gene from the many other genes in a genome
 Genome
• An organism’s complete set of genetic material
Nucleic Acid Hybridization
 Probes are used to identify one clone that hosts
a DNA fragment of interest among many other
clones in a DNA library
 Probe
• Short fragment of DNA labeled with a tracer
• Hybridizes with a specific nucleotide sequence
 Nucleic acid hybridization
• Base-pairing between DNA or RNA from different
sources
PCR
 PCR quickly mass-produces copies of a
particular DNA fragment for study
 Polymerase chain reaction (PCR)
• Uses primers and heat-resistant DNA polymerase
to rapidly generate many copies of a DNA
fragment
 Primer
• Short, single-strand of DNA designed to hybridize
with a DNA fragment
Steps in PCR
1. Starting material is mixed with DNA polymerase,
nucleotides and primers
2. Mixture is heated and cooled in cycles
• At high temperature, DNA unwinds
• At low temperature, primers base-pair with
template DNA
3. Taq polymerase synthesizes complementary
DNA strands on templates
Two Rounds of PCR
1 DNA template (blue) is mixed with
primers (red), nucleotides, and heattolerant Taq DNA polymerase.
2 When the mixture is heated, the
double-stranded DNA separates
into single strands. zhen it is
cooled, some of the primers
base-pair with the template DNA.
3 Taq polymerase begins DNA
synthesis at the primers, and
complementary strands of DNA
form on the single-stranded
templates.
4 The mixture is heated again,
and the double-stranded DNA
separates into single strands.
When it is cooled, some of the
primers base-pair with the
template DNA.
5 Taq polymerase begins DNA
synthesis at the primers, and
complementary strands of DNA
form on the single-stranded
templates.
Fig. 10-5, p. 183
1 DNA template (blue) is mixed with
primers (red), nucleotides, and heattolerant Taq DNA polymerase.
22When
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Whenititisiscooled,
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44The
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mixtureisisheated
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and
andthe
thedouble-stranded
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separates
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singlestrands.
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When
ititisiscooled,
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3 Taq polymerase begins DNA
synthesis at the primers, and
complementary strands of DNA
form on the single-stranded
templates.
5 Taq polymerase begins DNA
synthesis at the primers, and
complementary strands of DNA
form on the single-stranded
templates.
Stepped Art
Fig. 10-5, p. 183
Animation: Polymerase chain reaction
(PCR)
Animation: Formation of recombinant
DNA
Animation: Use of a radioactive probe
Animation: Base-pairing of DNA
fragments
Animation: How to make cDNA
Animation: Restriction enzymes
Animation: F2 ratios interaction
10.3 Studying DNA
 Short tandem repeats are multiple copies of a
short DNA sequence that follow one another
along a chromosome
 The number and distribution of short tandem
repeats, unique in each individual, is revealed
by electrophoresis as a DNA fingerprint
DNA Fingerprinting
 DNA fingerprinting is used in forensics, court
evidence, and other applications
 DNA fingerprint
• An individual’s unique array of short tandem
repeats
 Electrophoresis
• Used to separate DNA fragments by size
DNA Fingerprinting: A Forensic Case
Suspect 2
Female Cells
Semen
Size Reference
Boyfriend
Control DNA
Control DNA
Size Reference
Control DNA
Size Reference
Victim
Suspect 1
Size Reference
Evidence from
Crime Scene
Fig. 10-6, p. 184
The Human Genome Project
 Automated DNA sequencing and PCR enabled
scientists to sequence the more than 3 billion
bases of the human genome
 Sequencing
• Method of determining the order of nucleotides in
DNA
Sequencing a Fragment of DNA
 The order of colors is the order of DNA bases (A,
T, G, C)
Genomics
 Analysis of the human genome sequence is
yielding new information about human genes
and how they work
 Genomics
• The study of genomes (structural genomics,
comparative genomics)
Some Sequenced Genomes
Animation: Automated DNA sequencing
Animation: DNA fingerprinting
Video: ABC News: DNA mystery: Human
chimeras
Video: ABC News: Family ties: Paternity
testing
3D Animation: Gene sequencing
10.4 Genetic Engineering
 Recombinant DNA technology and genome
analysis are the basis of genetic engineering
 Genetic engineering is the directed alteration of
an individual’s genome, resulting in a genetically
modified organism (GMO)
 Genetic engineering
• Process by which deliberate changes are
introduced into an individual’s genome
Genetically Modified Microorganisms
 A transgenic organism carries a gene from a
different species
 Transgenic organisms are used in research,
medicine, and industry
 Transgenic bacteria and yeast produce
medically valuable proteins
Designer Plants
 Transgenic crop plants help farmers produce
food more efficiently
 Plants with modified or foreign genes are now
common in farm crops
Using the Ti plasmid
to Make a Transgenic Plant
1 An A. tumefaciens bacterium
has been engineered to contain a
Ti plasmid. The plasmid carries a
foreign gene.
2 The bacterium infects a plant
cell and transfers the Ti plasmid
into it. The plasmid DNA becomes
integrated into one of the cell’s
chromosomes.
3 The plant cell divides, and its
descendants form an embryo.
4 The embryo develops into
a transgenic plant.
5 The transgenic plant
expresses the foreign
gene. This tobacco plant is
expressing a gene
from a firefly.
Fig. 10-8, p. 187
Animation: Gene transfer using a Ti
plasmid
Genetically Modified Crops
 Bt gene confers insect resistance to corn
Biotech Barnyards
 Transgenic animals produce human proteins
 Animals that would be impossible to produce by
traditional breeding methods are being created
by genetic engineering
 Transgenic animals are used in research,
medicine, and industry
Transgenic Animals
Knockout Cells and Organ Factories
 Transgenic animals may one day provide a
source of organs and tissues for transplantation
into humans
 Xenotransplantation
• Transplant of an organ from one species to
another
Animation: Transferring genes into
plants
Video: ABC News: Cloned pooch
Video: ABC News: Mule clones
Video: ABC News: Glow-in-the-dark pigs
Video: ABC News: Cloned food approved
10.5 Genetically Modified Humans
 Genes can be transferred into a person’s cells to
correct a genetic defect or treat a disease
 However, the outcome of altering a person’s
genome remains unpredictable
 Gene therapy
• Transfer of a normal or modified gene into an
individual with the goal of treating a genetic
defect or disorder
Unpredictable Outcomes
 There are more than 15,000 serious genetic
disorders – gene therapy is the only real cure
 In some cases, gene therapy works – in other
cases it leads to death
• Inserting a virus-injected gene into a
chromosome can disrupt normal function and
cause cancer
• Severe allergic reaction to the viral vector can
cause death
One Successful Case of Gene Therapy
 Rhys Evans, born with a severe immune
disorder (SCID-X1) received a normal gene and
no longer lives in isolation
Getting Perfect
 Eugenics
• Idea of deliberately improving the genetic
qualities of the human race
 The potential benefits of genetically modifying
humans must be weighed against the potential
risks, including social implications
10.6 Impacts/Issues Revisited
 Golden rice with beta carotene was ready for
distribution in 2005 but is still not available for
human consumption – the biosafety experiments
required are too expensive for the public
humanitarian agency that developed it
Digging Into Data: Enhanced Spatial
Learning in Mice With Autism Mutation
Fig. 10-11a, p. 193
Fig. 10-11b, p. 193