2/24/12 Genetic Engineering

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14 Δεκ 2012 (πριν από 5 χρόνια και 23 μέρες)

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11.1 Restriction and Modification Enzymes


Genetic engineering
: using
in vitro

techniques
to alter genetic material in the laboratory


Basic techniques include



Restriction enzymes



Gel electrophoresis



Nucleic acid hybridization



Nucleic acid probes



Molecular cloning



Cloning vectors

© 2012 Pearson Education, Inc.

11.1
Restriction and Modification Enzymes


Restriction enzymes:

recognize specific DNA
sequences and cut DNA at those sites


Widespread among prokaryotes


Rare in eukaryotes


Protect prokaryotes from hostile foreign DNA
(e.g., viral genomes)


Essential for
in vitro

DNA manipulation

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2012
Pearson Education, Inc.

11.1 Restriction and Modification Enzymes


Three classes of restriction enzymes


Type II cleave DNA within their recognition
sequence and are most useful for specific DNA
manipulation (Figure
11.1
a)


Restriction enzymes recognize
inverted repeat

sequences (palindromes)


Typically
4

8
base pairs long;
Eco
RI recognizes a
6
-
base
-
pair sequence


Sticky ends

or
blunt ends

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2012
Pearson Education, Inc.

Figure
11.1
a

Single
-
stranded


獴楣歹


敮摳

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11.1
Restriction and Modification Enzymes


Restriction enzymes protect cell from invasion
from foreign DNA


Destroy foreign DNA


Must protect their own DNA from inadvertent
destruction

© 2012 Pearson Education, Inc.

11.1 Restriction and Modification Enzymes


Modification enzymes
: protect cell

s DNA
for restriction enzymes


Chemically modify nucleotides in restriction
recognition sequence


Modification generally consists of methylation
of DNA (Figure 11.1b)

©
2012
Pearson Education, Inc.

Figure
11.1
b

©
2012
Pearson Education, Inc.

11.1 Restriction and Modification Enzymes


Gel electrophoresis
: separates DNA molecules
based on size (Figure 11.2a)


Electrophoresis uses an electrical field to separate
charged molecules


Gels are usually made of agarose, a
polysaccharide


Nucleic acids migrate through gel toward the
positive electrode due to their negatively charged
phosphate groups


Gels can be stained with
ethidium bromide

and DNA can be visualized under UV light

(Figure 11.2b)

© 2012 Pearson Education, Inc.

Figure
11.2
a

©
2012
Pearson Education, Inc.

Figure
11.2
b

Size in base

pairs

A

B

C

D

5000

4000

3000

2000

1800

1000

500





卩穥 楮i扡獥

灡楲

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2012
Pearson Education, Inc.

11.1
Restriction and Modification Enzymes


The same DNA that has been cut with
different restriction enzymes will have
different banding patterns on an agarose gel


Size of fragments can be determined by
comparison to a standard


Restriction map
: a map of the location of
restriction enzyme cuts on a segment of DNA
(Figure 11.3)

© 2012 Pearson Education, Inc.

11.2
Nucleic Acid Hybridization



Nucleic acid hybridization
: base pairing of single
strands of DNA or RNA from two different sources
to give a hybrid double helix


Segment of single
-
stranded DNA that is used in
hybridization and has a predetermined identity is
called a
nucleic acid probe


Southern blot
: a hybridization procedure where
DNA is in the gel and probe is RNA or DNA


Northern blot
: RNA is in the gel

© 2012 Pearson Education, Inc.

Figure
11.4

© 2012 Pearson Education, Inc.

11.3
Essentials of Molecular Cloning


Molecular cloning
: isolation and incorporation of
a piece of DNA into a
vector

so it can be
replicated and manipulated


Three main steps of gene cloning (Figure
11.5
):

1.
Isolation and fragmentation of source DNA

2.
Insertion of DNA fragment into cloning vector

3.
Introduction of cloned DNA into host organism

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2012
Pearson Education, Inc.

Figure
11.5

Foreign DNA

Sticky

ends

Vector

Cloned

DNA

Introduction of recombinant

vector into a host

Cut with restriction

enzyme

Add vector cut

with same

restriction enzyme

Add DNA ligase to

form recombinant

molecules

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2012
Pearson Education, Inc.

11.3
Essentials of Molecular Cloning

1.
Isolation and fragmentation of source DNA


Source DNA can be genomic DNA, RNA, or
PCR
-
amplified fragments


Genomic DNA must first be restriction digested


© 2012 Pearson Education, Inc.

11.3
Essentials of Molecular Cloning

2.
Insertion of DNA fragment into cloning vector


Most vectors are derived from plasmids or
viruses


DNA is generally inserted
in vitro


DNA ligase
: enzyme that joins two DNA
molecules


Works with sticky or blunt ends

© 2012 Pearson Education, Inc.

11.3 Essentials of Molecular Cloning

3.
Introduction of cloned DNA into host organism


Transformation is often used to get recombinant
DNA into host


Some cells will contain desired cloned gene,
while other cells will have other cloned genes


Gene library:

mixture of cells containing a variety
of genes


Shotgun cloning
: gene libraries made by cloning
random genome fragments

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2012
Pearson Education, Inc.

Animation: Recombinant DNA

11.3
Essentials of Molecular Cloning


Essential to detect the correct clone


Initial screen: antibiotic resistance, plaque
formation


Often sufficient for cloning of PCR
-
generated
DNA sequences


If working with a heterogeneous gene library you
may need to look more closely

© 2012 Pearson Education, Inc.

Figure 11.6

Transformant colonies

growing on agar surface

X
-
ray film

Positive

colonies

Replica
-
plate onto

membrane filter

Partially lyse cells; add

specific antibody; add agent

to detect bound antibody in

radiolabeled form

Lyse bacteria and denature

DNA; add RNA or DNA

probe (radioactive); wash

out unbound radioactivity

Autoradiograph

to detect

radioactivity

© 2012 Pearson Education, Inc.


11.4
Molecular Methods for Mutagenesis


Synthetic DNA


Systems are available for de novo synthesis

of DNA


Oligonucleotides

of
100
bases can be made


Multiple oligonucleotides can be ligated together


Synthesized DNA is used for primers and probes,
and in site
-
directed mutagenesis

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2012
Pearson Education, Inc.

11.4 Molecular Methods for Mutagenesis


Conventional mutagens produce mutations at
random


Site
-
directed mutagenesis
: performed
in vitro
and introduces mutations at a precise location
(Figure 11.7)


Can be used to assess the activity of specific
amino acids in a protein


Structural biologists have gained significant
insight using this tool

© 2012 Pearson Education, Inc.

Figure 11.7

Single
-
stranded

DNA from M
13

phage

Base
-
pairing

with source

gene

Source

Clone and

select mutant

Clone into

single
-
stranded

vector

Add synthetic

oligonucleotide

with one base

mismatch

Extend single

strand with

DNA polymerase

Transformation

and selection

©
2012
Pearson Education, Inc.

11.4 Molecular Methods for Mutagenesis


Cassette mutagenesis

and
knockout mutations


DNA fragment can be cut, excised, and replaced
by a synthetic DNA fragment (DNA cassettes or
cartridges)


The process is known as
cassette mutagenesis



Gene disruption

is when cassettes are inserted into
the middle of the gene (Figure
11.8
)


Gene disruption causes knockout mutations



© 2012 Pearson Education, Inc.

Figure 11.8

Gene X

Eco
RI cut sites (

)

K慮慭a捩n 捡獳整瑥

Bam
HI

cut site

Linearized plasmid

Chromosome

Sites of recombination

Gene X knockout

Cut with
Eco
RI

and ligate

Cut with
Bam
HI and

transform into cell

with wild
-
type gene X

Recombination and selection

for kanamycin
-
resistant cells

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2012
Pearson Education, Inc.

Figure
11.9

© 2012 Pearson Education, Inc.

Figure 11.10

Target gene

Reporter gene

Gene fusion

Promoter

Promoter

Promoter

Coding sequence

Coding sequence

Cut and ligate

Reporter is expressed under

control of target gene promoter

Reporter

enzyme

Substrate

Colored product

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11.6 Plasmids as Cloning Vectors


Plasmids are natural vectors and have useful
properties as cloning vectors


Small size; easy to isolate DNA


Independent origin of replication


Multiple copy number; get multiple copies of
cloned gene per cell


Presence of selectable markers


Vector transfer carried out by chemical
transformation or electroporation

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2012
Pearson Education, Inc.

11.6 Plasmids as Cloning Vectors



pUC
19

is a common cloning vector (Figure
11.11
)


Modified ColE
1
plasmid


Contains ampicillin resistance and
lac
Z genes


Contains
polylinker

(
multiple cloning site
) within
lac
Z gene

© 2012 Pearson Education, Inc.

Figure 11.11

Ampicillin

resistance

Polylinker

Origin of

DNA replication

pUC19

2686 base pairs

lacI

lacZ


Order of restriction

enzyme cut sites in

polylinker

Apo
I
-

Eco
RI

Ban
II
-

Sac
I

Acc
651
-

Kpn
I

Ava
I
-

Bso
BI
-


Sma
I
-

Xma
I

Bam
HI

Xba
I

Acc
I
-

Hin
cII
-

Sal
I

Bsp
MI
-

Bfu
AI

Sbf
I

Pst
I

Sph
I

Hin
dIII

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2012
Pearson Education, Inc.

11.6 Plasmids as Cloning Vectors



Blue/white screening


Blue colonies do not have vector with foreign
DNA inserted


White colonies have foreign DNA inserted


Insertional inactivation
:
lac
Z gene is inactivated
by insertion of foreign DNA (Figure
11.12
)


Inactivated
lac
Z cannot process Xgal; blue color
does not develop

©
2012
Pearson Education, Inc.

Figure 11.12

AmpR

lacZ


Vector

Foreign DNA

Opened vector

Recyclized vector without insert

Vector plus foreign

DNA insert

Transformants blue

(

-
条污瑯s楤ise

a瑩te)

Transformants white

(

-
条污瑯s楤ise

楮i瑩te)

Digestion with restriction enzyme

Join with

DNA ligase

Transform into
Escherichia

coli

and select on ampicillin

plates containing Xgal

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2012
Pearson Education, Inc.

11.7 Hosts for Cloning Vectors


Ideal hosts should be


Capable of rapid growth in inexpensive medium


Nonpathogenic


Capable of incorporating DNA


Genetically stable in culture


Equipped with appropriate enzymes to allow
replication of the vector


Escherichia coli
,
Bacillus subtilis
,
Saccharomyces cerevisiae

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2012
Pearson Education, Inc.

Figure
11.13

Well
-
developed


genetics

Many strains


available

Best known


bacterium

Easily transformed

Nonpathogenic

Naturally secretes


proteins

Endospore formation


simplifies culture

Well
-
developed


genetics

Nonpathogenic

Can process mRNA


and proteins

Easy to grow

Potentially


pathogenic

Periplasm traps


proteins

Genetically unstable

Genetics less


developed than


in
E. coli

Plasmids unstable

Will not replicate


most bacterial


plasmids

Advantages

Disadvantages

Escherichia coli

Bacillus subtilis

Saccharomyces

cerevisiae

Bacteria

Eukaryote

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2012
Pearson Education, Inc.

11.5 Gene Fusions and Reporter Genes



Reporter genes


Encode proteins that are easy to detect and
assay (Figure
11.9
)


Examples:

lacZ
, luciferase, GFP

genes


Gene fusions


Promoters or coding sequences of genes of
interest can be swapped with those of reporter
genes to elucidate gene regulation under various
conditions

(Figure
11.10
)

© 2012 Pearson Education, Inc.

11.8
Shuttle Vectors and Expression
Vectors



Expression vectors
: allow experimenter to control
the expression of cloned genes (Figure
11.16
)


Based on transcriptional control


Allow for high levels of protein expression


Strong promoters


lac
,
trp
,
tac
,
trc
, lambda P
L


Effective transcription terminators are used to
prevent expression of other genes on the plasmid

©
2012
Pearson Education, Inc.

Figure
11.16

Ampicillin

resistance

Origin of

DNA replication

Polylinker

(cloning

site)

S/D

T
1

T
2

trc

promoter

lacO

lacI

© 2012 Pearson Education, Inc.

11.8
Shuttle Vectors and Expression
Vectors



In T
7
expression vectors, cloned genes are
placed under control of the T
7
promoter

(Figure
11.17
)


Gene for T
7
RNA polymerase present and under
control of easily regulated system (e.g.,
lac
)


T
7
RNA polymerase recognizes only T
7
promoters


Transcribes only cloned genes


Shuts down host transcription

© 2012 Pearson Education, Inc.

Figure 11.17

Induce
lac

promoter with

IPTG

T7 RNA

polymerase

Gene

product

Chromosome

Cloned


gene

T7

promoter

pET plasmid

Gene for

T7 RNA

polymerase

lac

operator

lac

promoter

lacl

©
2012
Pearson Education, Inc.

11.8
Shuttle Vectors and Expression
Vectors


mRNA produced must be efficiently translated
and there are problems with this always
happening


Bacterial ribosome binding sites are not present in
eukaryotic genomes


Differences in codon usage between organisms


Eukaryotic genes containing introns will not be
expressed properly in prokaryotes

© 2012 Pearson Education, Inc.