E. coli

Copyright
©
The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

CHAPTER 17

DNA Manipulation

•
Restriction endonucleases revolutionized
molecular biology

•
Enzymes that cleave DNA at specific sites

–
Used by bacteria against viruses

•
Restriction enzymes significant

–
Allow a form of physical mapping that was
previously impossible

–
Allow the creation of recombinant DNA
molecules (from two different sources)

2

•
3 types of restriction enzymes

•
Type I and III cleave with less precision
and are not used in manipulating DNA

•
Type II

–
Recognize specific DNA sequences

–
Cleave at specific site within sequence

–
Can lead to “sticky ends” that can be joined

•
Blunt ends can also be joined


3

4

5

•
DNA ligase

–
Joins the two fragments forming a stable DNA
molecule

–
Catalyzes formation of a phosphodiester bond
between adjacent phosphate and hydroxyl
groups of DNA nucleotides

–
Same enzyme joins Okazaki fragments on
lagging strand in replication

Gel Electrophoresis

•
Separate DNA fragments by size

•
Gel made of agarose or polyacrylamide

•
Submersed in buffer that can carry current

•
Subjected to an electrical field

•
Negatively
-
charged DNA migrates towards the
positive pole

•
Larger fragments move slower, smaller move
faster

•
DNA is visualized using fluorescent dyes

6

7

8

Transformation

•
Introduction of DNA from an outside
source into a cell

•
Natural process in many species

–
E. coli

does not

•
Temperature shifts can induce artificial
transformation in
E. coli


•
Transgenic organisms are all or part
transformed cells

9

Molecular Cloning

•
Clone
–

genetically identical copy

•
Molecular cloning
–

isolation of a specific
DNA sequence (usually protein
-
encoding)

–
Sometimes called gene cloning

•
The most flexible and common host for
cloning is
E. coli


–
Vector
–

carries DNA in host and can
replicate in the host

–
Each host
–
vector system has particular uses

10

Vectors

•
Plasmids

–
Small, circular chromosomes

–
Used for cloning small pieces of DNA

–
Selectable marker
–

allows presence of
plasmid to be easily identified

11

12

DNA Libraries

•
A collection of DNAs in a vector that taken
together represent the complex mixture of
DNA

•
Genomic library
–

representation of the
entire genome in a vector

–
Genome is randomly fragmented

–
Inserted into a vector

–
Introduced into host cells

–
Usually constructed in BACs

13

•
Southern blotting

–
Sample DNA is digested by restriction
enzymes and separated by gel
electrophoresis

–
Double
-
stranded DNA denatured into single
-
strands

–
Gel “blotted” with filter paper to transfer DNA

–
Filter is incubated with a labeled probe
consisting of purified, single
-
stranded DNA
corresponding to a specific gene


14

15

16

17

•
Northern blotting

–
mRNA is separated by electrophoresis and
then blotted onto the filter


•
Western blotting

–
Proteins are separated by electrophoresis and
then blotted onto the filter

–
Detection requires an antibody that can bind
to one protein

18

•
RFLP analysis

–
Restriction fragment length polymorphisms

–
Generated by point mutations or sequence
duplications

–
Restriction enzyme fragments are often not
identical in different individuals

–
Can be detected by Southern blotting

19

20

•
DNA fingerprinting

–
Identification technique used to detect
differences in the DNA of individuals

–
Population is polymorphic for these markers

–
Using several probes, probability of identity
can be calculated or identity can be ruled out

–
First used in a U.S. criminal trial in 1987

•
Tommie Lee Andrews was found guilty of rape

–
Also used to identify remains

21

22

•
DNA sequencing

–
Determination of actual
base sequence of DNA

–
Basic idea is nested
fragments

–
Each begin with the same
sequence and end in a
specific base

–
By starting with the
shortest fragment, one
can then read the
sequence by moving up
the ladder


•
Automated DNA sequencing

–
Enzymatic technique is powerful but is labor
-
intensive and time
-
consuming

–
Automation made sequencing faster and
more practical

–
Fluorescent dyes are used instead of
radioactive labels

–
Reaction is done in one tube

–
Data are assembled by a computer

23

24

•
Fundamentally new method for DNA sequencing

–
DNA is cleaved into smaller pieces

–
Both ends are ligated to adapters that are
complementary to specific primers

–
DNA fragments are injected into a flow cell

–
Each of 7 channels contains a solid substrate with
primers that complement the ligated ends of the DNA
fragments

–
Uses fluorescent tag on dNTPs

–
Camera records colors after each round of extension

25

26

Copyright © The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

NH
2

O

P

O
–

–
O

O

CH
2

5
´

4
´

3
´

2
´

1
´

OH

N

N

N

O

A

Reversible terminator

A

A

T

T

G

G

C

A

T

G

C

C

g.

h.

First round of

synthesis with

labeled dNTPs

Image capture for each

round of synthesis

27

Copyright © The McGraw
-
Hill Companies, Inc. Permission required for reproduction or display.

b: © 2007, Illumina Inc. All rights reserved

Adapter

Adapter

Attached

Clusters

Adapters

a.

b.

c.

d.

e.

f.

Flow cell

1 cm

DNA

DNA fragment

Dense primer lawn

in flow cell

35 cycles

of bridge

amplification

Bridge

amplification

with unlabeled

dNTPs

Free end

binds to

primer

Fragments

become

double
-

stranded

Free

terminus

Denature

double
-

stranded

molecules

Attached

•
Polymerase chain reaction (PCR)

–
Developed by Kary Mullis

•
Awarded Nobel Prize

–
Allows the amplification of a small DNA
fragment using primers that flank the region

–
Each PCR cycle involves three steps:

1.
Denaturation (high temperature)

2.
Annealing of primers (low temperature)

3.
DNA synthesis (intermediate temperature)

–
Taq polymerase

28

29



After 20 cycles, a
single fragment
produces over one
million (2
20
) copies!

30

•
Applications of PCR

–
Allows the investigation of minute samples of
DNA

–
Forensics
–

drop of blood, cells at base of a
hair

–
Detection of genetic defects in embryos by
analyzing a single cell

–
Analysis of mitochondrial DNA from early
human species

31

Medical Applications

•
Medically important proteins can be
produced in bacteria

–
Human insulin

–
Interferon

–
Atrial peptides

–
Tissue plasminogen activator

–
Human growth hormone

–
Problem has been purification of desired
proteins from other bacterial proteins

32

Genetically engineered mouse

with human growth hormone


33

•
Vaccines

–
Subunit vaccines

•
Genes encoding a part of the protein coat are
spliced into a fragment of the vaccinia (cowpox)
genome

•
Injection of harmless recombinant virus leads to
immunity

–
DNA vaccines

•
Depend on the cellular immune response (not
antibodies)

34

35

•
Gene therapy

–
Adding a functional copy of a gene to correct
a hereditary disorder

–
Severe combined immunodeficiency disease
(SCID) illustrates both the potential and the
problems

•
On the positive side, 15 children treated
successfully are still alive

•
On the negative side, three other children treated
have developed leukemia (due to therapy)

36

Agricultural Applications

•
Ti (tumor
-
inducing) plasmid

–
Most used vector for plant genetic
engineering

–
Obtained from
Agrobacterium tumefaciens,
which normally infects broadleaf plants

–
Part of the Ti plasmid integrates into the plant
DNA and other genes can be attached to it

–
However, bacterium does not infect cereals
such as corn, rice, and wheat

37

38

39

•
Other methods of gene insertion

–
Gene guns

•
Uses bombardment with tiny gold particles coated
with DNA

•
Possible for any species

•
Copy number of inserted genes cannot be
controlled

–
Modification of
Agrobacterium
system

–
Use of other bacteria like
Agrobacterium

40

•
Herbicide resistance

–
Broadleaf plants have been engineered to be
resistant to the herbicide glyphosate

–
Benefits

•
Crop resistant to glyphosate would not have to be
weeded

•
Single herbicide instead of many types

•
Glyphosate breaks down in environment

–
In the United States, 90% of soy currently
grown is GM soy

41

•
Bt crops

–
Insecticidal proteins have been transferred
into crop plants to make them pest
-
resistant

–
Bt toxin from
Bacillus thuringiensis


–
Use of Bt maize is the second most common
GM crop globally

•
Stacked crops

–
Both glyphosate
-
resistant and Bt
-
producing

42

•
Golden rice

–
Rice that has been genetically modified to
produce
b
-
carotene (provitamin A)

–
Converted in the body to vitamin A

–
Interesting for 2 reasons

•
Introduces a new biochemical pathway in tissue of
the transgenic plants

•
Could not have been done by conventional
breeding as no rice cultivar known produces these
enzymes in endosperm

–
Available free with no commercial
entanglements

43

44

•
Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions

–
Crop safety for human consumption

–
Movement of genes into wild relatives

•
No evidence so far but it is not impossible

45

•
Biopharming

–
Transgenic plants are used to produce
pharmaceuticals

–
1990
–

Human serum albumin produced in
genetically engineered tobacco and potato
plants

–
In development

•
Recombinant subunit vaccines against Norwalk
and rabies viruses

•
Recombinant monoclonal antibodies against tooth
decay
-
causing bacteria

46

•
Transgenic animal technology has not
been as successful as that in plants

•
Molecular techniques combined with the
ability to clone domestic animals could
produce improved animals for
economically desirable traits

•
Main use thus far has been engineering
animals to produce pharmaceuticals in
milk (also biopharming)

47