CH9

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Feb 21, 2013 (4 years and 6 months ago)

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Copyright © 2013 Pearson Education, Inc.

Lectures prepared by Christine L. Case

Chapter 9

Biotechnology
and

DNA Technology

© 2013 Pearson Education, Inc.

Lectures prepared by Christine L. Case

© 2013 Pearson Education, Inc.

© 2013 Pearson Education, Inc.

9
-
1

Compare and contrast biotechnology, genetic
modification, and recombinant DNA technology.

9
-
2

Identify the roles of a clone and a vector in
making recombinant DNA.




Introduction to Biotechnology

Learning Objectives

© 2013 Pearson Education, Inc.

Biotechnology and Recombinant DNA


Biotechnology
: the use of microorganisms,
cells, or cell components to make a product


Foods, antibiotics, vitamins, enzymes


Recombinant DNA (rDNA) technology
:
insertion or modification of genes to produce
desired proteins

© 2013 Pearson Education, Inc.

Biotechnology and Recombinant DNA


Vector
: self
-
replicating DNA used to carry the
desired gene to a new cell


Clone
: population of cells arising from one cell; each
carries the new gene

© 2013 Pearson Education, Inc.

Figure 9.1 A Typical Genetic Modification Procedure.

bacterium

recombinant DNA

(plasmid)

transformed
bacterium

Plasmid

Bacterial

cromosome

Vector, such as a
plasmid, is isolated.

DNA containing gene of interest from a
different species is cleaved by an enzyme

into fragments.

Desired gene is selected and inserted into plasmid.

Plasmid is taken up by a cell, such
as a bacterium.

Cells with gene of interest are cloned
with either of two goals in mind.

Create and harvest
copies of a gene.

Create and harvest

protein products of a gene.

Plasmid

RNA

Protein product

or

Gene encoding
protein for pest
resistance is inserted
into plant cells.

Gene encoding
degradative enzyme to
clean up toxic waste is
inserted into bacterial
cells.

Amylase, cellulase, and
other enzymes prepare
fabrics for clothing
manufacture.

Human growth
hormone treats
stunted growth.

DNA containing

gene of interest

© 2013 Pearson Education, Inc.

Table 9.2 Some Pharmaceutical Products of rDNA

© 2013 Pearson Education, Inc.

Table 9.3 Some Agriculturally Important Products of rDNA Technology

© 2013 Pearson Education, Inc.

Check Your Understanding


Differentiate biotechnology and recombinant DNA
technology.
9
-
1


In one sentence, describe how a vector and clone
are used.
9
-
2



© 2013 Pearson Education, Inc.

9
-
3

Compare selection and mutation.

9
-
4

Define
restriction enzymes
, and outline how they
are used to make recombinant DNA.

9
-
5

List the four properties of vectors.

9
-
6

Describe the use of plasmid and viral vectors.

9
-
7

Outline the steps in PCR, and provide an
example of its use.




Tools of Biotechnology

Learning Objectives

© 2013 Pearson Education, Inc.

Selection and Mutation


Selection
: culture a naturally occurring microbe that
produces the desired product


Mutation
: mutagens cause mutations that might
result in a microbe with a desirable trait


Site
-
directed mutagenesis
: change a specific DNA
code to change a protein


Select and culture a microbe with the desired
mutation

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Restriction Enzymes


Cut specific sequences of DNA


Destroy bacteriophage DNA in bacterial cells


Cannot digest (host) DNA with methylated cytosines

ANIMATION: Recombinant DNA Technology

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Table 9.1 Selected Restriction Enzymes Used in rDNA Technology

© 2013 Pearson Education, Inc.

Figure 9.2 The role of a restriction enzyme in making recombinant DNA.

DNA

Cut

Cut

Recognition sites

Cut

Cut

Sticky end

Restriction enzyme

cuts (red arrows)

double
-
stranded DNA at its
particular recognition sites,
shown in blue.

These cuts produce a DNA
fragment with two stick
ends.

When two such fragments
of DNA cut by the same
restriction enzyme come
together, they can join by
base pairing.

The joined fragments will usually
form either a linear molecule or

a circular one, as shown here for
a plasmid. Other combinations of
fragments can also occur.

The enzyme DNA ligase is used to
unite the backbones of the two
DNA fragments, producing a
molecule of recombinant DNA.

Recombinant DNA

DNA from another
source, perhaps

a plasmid, cut

with the same
restriction enzyme.

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Vectors


Carry new DNA to desired cell


Shuttle vectors

can exist in several different
species


Plasmids and viruses can be used as vectors

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pUC19

amp
R

lacZ

Hin
dIII

Bam
HI

Eco
RI

ori

Figure 9.3 A plasmid used for cloning.

© 2013 Pearson Education, Inc.

© 2013 Pearson Education, Inc.

Polymerase Chain Reaction (PCR)


To make multiple copies of a piece of DNA
enzymatically


Used to:


Clone DNA for recombination


Amplify DNA to detectable levels


Sequence DNA


Diagnose genetic disease


Detect pathogens

ANIMATION PCR: Components

ANIMATION PCR: Overview

© 2013 Pearson Education, Inc.

Incubate target DNA

at 94
°
C for 1 minute to

separate the strands.

Add primers, nucleotides
(deoxynucleotides, dNTP), and
DNA polymerase.

Primers attach to single
-
stranded DNA during
incubation at 60
°
C for 1
minute.

Incubate at 72
°
C for 1 minute;
DNA polymerase copies the
target DNA at this temperature.

Repeat the cycle of

heating and cooling

to make two more

copies of target DNA.

Target DNA

Primer

DNA

polymerase

dNTP

First cycle

Second cycle

Figure 9.4 The polymerase chain reaction.

© 2013 Pearson Education, Inc.

© 2013 Pearson Education, Inc.

Check Your Understanding


How are selection and mutation used in
biotechnology?
9
-
3


What is the value of restriction enzymes in
recombinant DNA technology?
9
-
4


What criteria must a vector meet?
9
-
5


Why is a vector used in recombinant DNA
technology?
9
-
6


For what is each of the following used in PCR:
primer, DNA polymerase, 94
°
C?
9
-
7

© 2013 Pearson Education, Inc.

9
-
8

Describe five ways of getting DNA into a cell.

9
-
9

Describe how a genomic library is made.

9
-
10

Differentiate cDNA from synthetic DNA.

9
-
11

Explain how each of the following is used to
locate a clone: antibiotic
-
resistance genes, DNA
probes, gene products.

9
-
12

List one advantage of modifying each of the
following:
E. coli
,
Saccharomyces cerevisiae
,
mammalian cells, plant cells.





Techniques of Genetic Modification

Learning Objectives

© 2013 Pearson Education, Inc.

Inserting Foreign DNA into Cells


DNA can be inserted into a cell by:


Electroporation


Transformation


Protoplast fusion


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Figure 9.5b Protoplast fusion.

Algal protoplasts fusing

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Figure 9.5a Protoplast fusion.

Process of protoplast fusion

Chromosome

Plasma membrane

Cell wall

Bacterial cell walls are
enzymatically digested,
producing protoplasts.

In solution, protoplasts are treated
with polyethylene glycol.

Protoplasts fuse.

Segments of the two
chromosomes recombine.

Recombinant cell grows new
cell wall.

Recombinant cell

Protoplasts

Bacterial
Cells

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Inserting Foreign DNA into Cells


DNA can be inserted into a cell by:


Gene gun


Microinjection

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Figure 9.6 A gene gun, which can be used to insert DNA
-
coated “bullets” into a cell.

© 2013 Pearson Education, Inc.

Figure 9.7 The microinjection of foreign DNA into an egg.

© 2013 Pearson Education, Inc.

© 2013 Pearson Education, Inc.

Obtaining DNA


Genomic libraries

are made of pieces of an entire
genome stored in plasmids or phages

© 2013 Pearson Education, Inc.

Genome to be stored
in library is cut up with
restriction enzyme

Recombinant
plasmid

Host

cell

Recombinant
phage DNA

Phage cloning
vector

OR

Bacterial
clone

Phage
clone

Plasmid library

Phage library

Figure 9.8 Genomic libraries.

© 2013 Pearson Education, Inc.

Obtaining DNA


Complementary DNA (cDNA)

is made from mRNA
by reverse transcriptase

© 2013 Pearson Education, Inc.

A gene composed of exons and
introns is transcribed to RNA by
RNA polymerase.

Processing enzymes in the
nucleus remove the intron
-
derived RNA and splice
together the exon
-
delivered
RNA into mRNA.

mRNA is isolated from the
cell, and reverse
transcriptase is added.

First strand
of DNA is
synthesized.

The mRNA is digested by
reverse transcriptase.

DNA polymerase is added to
synthesize second strand

of DNA.

DNA

RNA

transcript

mRNA

DNA strand

being synthesized

cDNA of

gene without

introns

Exon

Exon

Exon

Intron

Intron

Nucleus

Cytoplasm

Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene.

© 2013 Pearson Education, Inc.

Obtaining DNA


Synthetic DNA

is made by a DNA synthesis
machine

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Figure 9.10 A DNA synthesis machine.

© 2013 Pearson Education, Inc.

Figure 9.11 Blue
-
white screening, one method of selecting recombinant bacteria.

Plasmid DNA and foreign DNA are both cut
with the same restriction enzyme. The
plasmid has the genes for lactose
hydrolysis (the
lacZ

gene encodes the
enzyme

-
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牥獩獴慮捥r

Foreign DNA will insert into the
lacZ

gene. The bacterium receiving the
plasmid vector will not produce the
enzyme

-
条污捴潳楤gs攠楦i景牥楧渠䑎D
桡猠扥敮b楮獥牴敤i楮瑯i瑨攠灬慳浩搮


The recombinant plasmid is introduced
into a bacterium, which becomes
ampicillin resistant.

Foreign DNA will insert into the
lacZ

gene.
The bacterium receiving the plasmid vector
will not produce the enzyme

-
条污捴潳楤gs攠楦i景牥楧渠䑎D 桡猠扥敮b
楮獥牴敤i楮瑯i瑨攠灬慳浩搮p

Only bacteria that picked up the plasmid
will grow in the presence of ampicillin.

Bacteria that hydrolyze X
-
gal produce
galactose and an indigo compound.

The indigo turns the colonies blue.

Bacteria that cannot hydrolyze X
-
gal
produce white colonies.


-
galactosidase
gene (
lacZ)

Ampicillin
-
resistance
gene (
amp
R
)

Restriction
site

Restriction sites

Foreign DNA

Recombinant

plasmid

Bacterium

Colonies
with foreign
DNA

Plasmid

© 2013 Pearson Education, Inc.

Make replica of master plate on
nitrocellulose filter.

Treat filter with detergent (SDS)
to lyse bacteria.

Treat filter with sodium hydroxide
(NaOH) to separate DNA

into single strands.

Add labeled probes.

Probe will hybridize with desired
gene from bacterial cells.

Wash filter to remove unbound
probe.

Compare filter with replica of
master plate to identify colonies
containing gene of interest.

Master plate with colonies of bacteria
containing cloned segments of foreign genes.

Nitrocellulose filter

Strands of

bacterial DNA

Fluorescence
labeled probes

Bound DNA probe

Gene of interest

Single
-
stranded DNA

Colonies containing
genes of interest

Replica plate

Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest.

© 2013 Pearson Education, Inc.

Making a Product


E. coli


Used because it is easily grown and its genomics are
known


Need to eliminate endotoxin from products


Cells must be lysed to get product

© 2013 Pearson Education, Inc.

Figure 9.13
E. coli

genetically modified to produce gamma interferon, a human protein that promotes an
immune response.

© 2013 Pearson Education, Inc.

© 2013 Pearson Education, Inc.

Making a Product


Saccharomyces cerevisiae


Used because it is easily grown and its genomics are
known


May express eukaryotic genes easily


Plant cells and whole plants


May express eukaryotic genes easily


Plants are easily grown


Mammalian cells


May express eukaryotic genes easily


Harder to grow







© 2013 Pearson Education, Inc.

Check Your Understanding


Contrast the five ways of putting DNA into a cell.
9
-
8


What is the purpose of a genomic library?
9
-
9


Why isn’t cDNA synthetic?
9
-
10


How are recombinant clones identified?
9
-
11


What types of cells are used for cloning rDNA?
9
-
12


© 2013 Pearson Education, Inc.

Applications of rDNA

9
-
13

List at least five applications of rDNA
technology.

9
-
14

Define RNAi.

9
-
15

Discuss the value of genome projects.

9
-
16

Define the following terms:
random shotgun
sequencing
,

bioinformatics
,

proteomics
.





Learning Objectives

© 2013 Pearson Education, Inc.

Therapeutic Applications


Human enzymes

and other proteins


Subunit vaccines


Nonpathogenic viruses carrying genes for
pathogen’s antigens as
DNA vaccines


Gene therapy

to replace defective or missing genes

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Nucleus

Cytoplasm

DNA

RNA

transcript

mRNA

An abnormal gene,
cancer gene, or virus
gene is transcribed in a
host cell.

siRNA binds

mRNA.

RISC breaks
down the RNA
complex.

No protein
expression occurs.

siRNA

Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.

© 2013 Pearson Education, Inc.

Figure 9.15 Shotgun sequencing.

Isolate DNA.

Fragment DNA
with restriction
enzymes.

Clone DNA

in a bacterial
artificial
chromosome
(BAC).

Sequence DNA fragments.

Assemble sequences.

Edit
sequences;
fill in gaps.

Constructing a gene library

Random sequencing

Closure phase

BAC

© 2013 Pearson Education, Inc.

The Human Genome Project


Nucleotides have been sequenced


Human Proteome Project

may provide diagnostics
and treatments


Reverse genetics
: block a gene to determine its function

© 2013 Pearson Education, Inc.

Check Your Understanding


Explain how rDNA technology can be used to treat
disease and to prevent disease.
9
-
13


What is gene silencing?
9
-
14


How are shotgun sequencing, bioinformatics, and
proteomics related to the Human Genome Project?
9
-
15, 9
-
16



© 2013 Pearson Education, Inc.

Applications of rDNA


9
-
17

Diagram the Southern blotting procedure, and
provide an example of its use.

9
-
18

Diagram DNA fingerprinting, and provide an
example of its use.

9
-
19

Outline genetic engineering with
Agrobacterium
.





Learning Objectives

© 2013 Pearson Education, Inc.

Scientific Applications


Understanding DNA


Sequencing organisms’ genomes


DNA fingerprinting for identification

© 2013 Pearson Education, Inc.

Figure 9.17 DNA fingerprints used to track an infectious disease.

E. coli

isolates from

patients whose

infections were

not juice related

E. coli

isolates from

patients who drank
contaminated juice

Apple juice
isolates

© 2013 Pearson Education, Inc.

DNA containing the gene of interest is extracted
from human cells and cut into fragments by
restriction enzymes.

The fragments are separated according to size by
gel electrophoresis. Each band consists of many
copies of a particular DNA fragment. The bands
are invisible but can be made visible by staining.

The DNA bands are transferred to a nitrocellulose
filter by blotting. The solution passes through the
gel and filter to the paper towels by capillary
action.

This produces a nitrocellulose filter with DNA
fragments positioned exactly as on the gel.

The filter is exposed to a labeled probe for a
specific gene. The probe will base
-
pair (hybridize)
with a short sequence present on the gene.

The fragment containing the gene of interest is
identified by a band on the filter.

Restriction
enzyme

Gene of
interest

Larger

Smaller

Gel

Human
DNA
fragments

Paper
towels

Salt
solution

Gel

Sponge

Nitrocellulose
filter

Labeled probes

Sealable
plastic bag

Nitrocellulose
filter

DNA
transferred
to filter

Gel

Figure 9.16 Southern blotting.

© 2013 Pearson Education, Inc.

Forensic Microbiology


PCR


Primer for a specific organism will cause application
if that organism is present


Real
-
time PCR
: newly made DNA is tagged with a
fluorescent dye; the levels of fluorescence can be
measured after every PCR cycle


Reverse
-
transcription (RT
-
PCR)
: reverse
transcriptase makes DNA from viral RNA or mRNA

© 2013 Pearson Education, Inc.

Forensic Microbiology


Differs from medicine because it requires:


Proper evidence collection


Establishing chain of custody


Rape conviction


Tracing HIV to a physician who injected it


Anthrax in U.S. Mail


© 2013 Pearson Education, Inc.

Norovirus Outbreak


Are the outbreaks related?


What is the source?

© 2013 Pearson Education, Inc.

Chapter 9, unnumbered figure A, page 265.

School
meal
served

Initial reports
of community
cases

© 2013 Pearson Education, Inc.

Norovirus Outbreak


RT
-
PCR with a norovirus primer

© 2013 Pearson Education, Inc.

Chapter 9, unnumbered figure B, page 265.

213

1

8

3

4

5

6

2

7

© 2013 Pearson Education, Inc.

Nanotechnology


Bacteria can make molecule
-
sized particles

© 2013 Pearson Education, Inc.

Figure 9.18
Bacillus
cells growing on selenium form chains of elemental selenium.

© 2013 Pearson Education, Inc.

Using
Agrobacterium


Bt toxin


Herbicide resistance


Suppression of genes


Antisense DNA


Nutrition


Human proteins

© 2013 Pearson Education, Inc.

Crown gall

Figure 9.19 Crown gall disease on a rose plant.

© 2013 Pearson Education, Inc.

Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants.

Agrobacterium tumefaciens
bacterium

Restriction
cleavage
site

T
-
DNA

Inserted T
-
DNA
carrying foreign
gene

Ti
plasmid

Recombinant
Ti plasmid

The plasmid is removed

from the bacterium, and

the T
-
DNA is cut by a
restriction enzyme.

Foreign DNA is cut

by the same enzyme.

The foreign DNA is
inserted into the T
-
DNA
of the plasmid.

The plasmid

is reinserted

into a bacterium.

The bacterium is
used to insert the

T
-
DNA carrying the
foreign gene into the
chromosome of a
plant cell.

The plant cells
are grown in
culture.

A plant is generated from a cell
clone. All of its cells carry the
foreign gene and may express

it as a new trait.

© 2013 Pearson Education, Inc.

Check Your Understanding


What is Southern blotting?
9
-
17


Why do RFLPs result in a DNA fingerprint?
9
-
18


Of what value is the plant pathogen
Agrobacterium
?
9
-
19



© 2013 Pearson Education, Inc.

Safety Issues and Ethics of Using rDNA

9
-
20

List the advantages of, and problems associated
with, the use of genetic modification techniques.





Learning Objective

© 2013 Pearson Education, Inc.

Safety Issues and Ethics of Using rDNA


Need to avoid accidental release


Genetically modified crops must be safe for
consumption and for the environment


Who will have access to an individual’s genetic
information?

© 2013 Pearson Education, Inc.

Check Your Understanding


Identify two advantages and two problems
associated with genetically modified organisms.
9
-
20