Chapter 10

Biotechnology

Chapter 10

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

Fig. 10
-
2, p. 181

restriction
enzyme (cut)

DNA ligase
(paste)

1

A restriction enzyme
recognizes a specific base
sequence in DNA (green
boxes) from two sources.

2

The enzyme cuts DNA
from both sources into
fragments that have
sticky ends.

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.

mix

Making Recombinant DNA

Fig. 10
-
3, p. 181

Bam

Hl

Pst

l

Sph

l

Kpn

l

Eco

RI

Sal

l

Acc

l

Xho

l

Xba

l

Sac

l

Bst

XI

Not

l

Cloning Vector 3.85 kb

Plasmid

The use of mRNA for the
Identification of DNA

•
mRNA sequence comes from specific
regions of DNA (Genes)

•
mRNA sequence is used to make proteins
and defines the physical/behavioral
characteristics of the organism

•
Therefore we use mRNA to identify active
regions of DNA

•
Use mRNA sequence and base pairing
rules to identify DNA original sequence

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

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

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

2

When the mixture is heated, the
double
-
stranded DNA separates into
single strands. When it is cooled,
some of the primers base
-
pair with the
template DNA.

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.

Fig. 10
-
5, p. 183

1

DNA template (
blue
) is mixed with
primers (
red
), nucleotides, and heat
-
tolerant
Taq

DNA polymerase.

Stepped Art

3

Taq

polymerase begins DNA
synthesis at the primers, and
complementary strands of DNA
form on the single
-
stranded
templates.

2

When the mixture is heated, 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.

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.

Two Rounds of PCR

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

Fig. 10
-
6, p. 184

Evidence from
Crime Scene

Size Reference

Control DNA

Size Reference

Victim

Suspect 1

Suspect 2

Female Cells

Semen

Size Reference

Boyfriend

Control DNA

Control DNA

Size Reference

DNA Fingerprinting: A Forensic Case

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

Fig. 10
-
8, p. 187

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.

Using the Ti plasmid

to Make a Transgenic Plant

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