Transcript Brooker Chapter 18 - Volunteer State Community College

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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 18 Part 1
RECOMBINANT
DNA TECHNOLOGY
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18-4
Cloning Experiments Involve
Chromosomal and Vector DNA

Cloning experiments usually involve two kinds of
DNA molecules

Chromosomal DNA or cDNA


Serves as the source of the DNA segment of interest
Vector DNA

Serves as the carrier of the DNA segment that is to be cloned
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18-5

The cell that harbors the vector is called the host cell


When a vector is replicated inside a host cell, the DNA that
it carries is also replicated
The vectors commonly used in gene cloning were
originally derived from two natural sources

1. Plasmids
2. Viruses

Commercially available plasmids have selectable markers



Typically, genes conferring antibiotic resistance to the host cell
Table 18.2 provides a general description of several
vectors used to clone small segments of DNA
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18-6
Cloning Experiments Involve
Enzymes that Cut and Paste DNA

Insertion of chromosomal DNA into a vector
requires the cutting and pasting of DNA fragments

The enzymes used to cut DNA are known as
restriction endonucleases or restriction enzymes


These bind to specific DNA sequences and then cleave
the DNA at two defined locations, one on each strand
Figure 18.1 shows the action of a restriction
endonuclease
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18-8
To be continued 
Figure 18.1
18-9

Restriction enzymes are made naturally by many
species of bacteria


They protect bacterial cells from invasion by foreign DNA,
particularly that of bacteriophage
Currently, several hundred different restriction
enzymes are available commercially

Table 18.3 gives a few examples
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18-10
18-11

Restriction enzymes bind to specific DNA sequences

These are typically palindromic

For example, the EcoRI recognition sequence is
5’ GAATTC 3’
3’ CTTAAG 5’


Some restriction enzymes digest DNA into
fragments with “sticky ends”
Other restriction enzymes generate blunt ends
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18-12
Figure 19-2
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This interaction is not stable because
it involves only a few hydrogen bonds
To establish a permanent connection, the
sugar-phosphate backbones of the two DNA
fragments must be covalently linked
Add DNA ligase which
covalently links the
DNA backbones
A recombinant
DNA molecule
Figure 18.1
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The Steps in Gene Cloning

The general strategy followed in a typical cloning
experiment is outlined in Figure 18.2

The procedure shown seeks to clone the human
b-globin gene into a plasmid vector

The vector carries two important genes


ampR  Confers antibiotic resistance to the host cell
lacZ  Encodes b-galactosidase

Provides a means by which bacteria that have picked up the cloned
gene can be identified
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18-14
Figure 19-6
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This is termed
a hybrid vector
Figure 18.2
Note: In this case, the b-globin
gene was inserted into the plasmid
It is also possible for any other
DNA fragment to be inserted into
the plasmid
And it is possible for the plasmid
to circularize without an insert
This is called a recircularized
vector
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This step of the procedure
is termed transformation.
Cells that are able to take
up DNA are called
competent cells
Figure 18.2
18-16





Nonrecombinant: recircularized
Recombinant: vector plus inserted cloned gene
Selection for vector: ampicillin resistance
Selection for recombinant vs. nonrecombinant
vector: b-galactosidase activity
Selection for for gene of interest?
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18-17

The growth media contains two relevant compounds:

IPTG (isopropyl-b-D-thiogalactopyranoside)


X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside)


A colorless compound that is cleaved by b-galactosidase into a
blue dye
The color of bacterial colonies will therefore depend on
whether or not the b-galactosidase is functional



A lactose analogue that can induce the lacZ gene
If it is, the colonies will be blue
If not, the colonies will be white
In this experiment


Bacterial colonies with recircularized vectors form blue colonies
While those with hybrid vectors form white colonies
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18-18

The net result of gene cloning is to produce an
enormous amount of copies of a gene


During transformation, a single bacterial cell usually
takes up a single copy of the hybrid vector
Amplification of the gene occurs in two ways:



1. The vector gets replicated by the host cell many times
2. The bacterial cell divides approximately every 30 minutes
Recombinant DNA technology is not only used to
clone genes

Sequences such as telomeres, centromeres and highly
repetitive sequences can be cloned as well
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18-19
Experiment 18A: The First Gene
Cloning Experiment

This was accomplished by Stanley Cohen, Annie
Chang, Herbert Boyer, and Robert Helling in 1973

Several important discoveries led to their ability to
clone a gene



DNA ligase covalently links DNA fragments together
EcoRI produces sticky ends when digesting DNA
Cohen et al realized that it is possible to create
recombinant DNA molecules using

EcoRI then DNA ligase
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18-20

They chose a plasmid for
their vector

The plasmid was designated
pSC101

The insertion of the gene will
occur at the lone EcoRI site

As source of the gene, they
obtained a second plasmid


Tetracycline
resistance
They called it pSC102
One of the three EcoRI
fragments is expected to
carry the KanR gene
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Kanamycin
resistance
18-21
The Hypothesis

A piece of DNA carrying a gene can be inserted
into a plasmid vector using recombinant DNA
techniques

If this recombinant plasmid is introduced into a
bacterial host cell, it will be replicated and transmitted
to daughter cells, producing many copies of the
recombinant plasmid
Testing the Hypothesis

Refer to Figure 18.3
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18-22
Figure 18.3
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Figure 18.3
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Figure 18.3
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Figure 18.3
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Figure 18.3
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Single peak
A single plasmid
with intermediate
density; NOT a
mixture of two
plasmids
The Data
Density
Gradient
Centrifugation
Control Experiment
pSC102
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pSC101
18-28
The Data
Gel
Electrophoresis
This band
corresponds
to pSC101
This band is
also found in
pSC102
18-29

The recombinant plasmid is shown here

This experiment showed it is possible to create recombinant
DNA molecules and to propagate them in bacterial cells

This hallmark achievement ushered in the era of gene cloning
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18-30
cDNA

To clone DNA, one can start with a sample of RNA

The enzyme reverse transcriptase is used


DNA that is made from RNA is called complementary
DNA (cDNA)


Uses RNA as a template to make a complementary strand of DNA
It could be single- or double-stranded
Synthesis of cDNA is presented in Figure 18.4
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18-31
polyA tail
Figure 18.4
18-32

From a research perspective, an important
advantage of cDNA is that it lacks introns

This has two ramifications

1. It allows researchers to focus their attention on the
coding sequence of a gene

2. It allows the expression of the encoded protein
Especially, in cells that would not splice out the introns properly
(e.g., a bacterial cell)

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18-33
Gel electrophoresis
Nucleic acid electrophoresis separates
DNA and RNA fragments by size
 smaller fragments migrate at a faster
rate through a gel than large fragments.

Figure 10-27
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Restriction Mapping

Sometimes, it is necessary to obtain smaller clones
from a large chromosomal DNA insert

This process is termed subcloning

Cloning and subcloning require knowledge of the
locations of restriction enzyme sites in vectors and
hybrid vectors

A common approach to examine the locations of
restriction sites is known as restriction mapping

Figure 18.5 outlines the restriction mapping of a bacterial
plasmid, pBR322
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18-34
Figure 18.5
18-35
Used for
fragment
size
comparison
Figure 18.5
18-36

The restriction map can be deduced by comparing the sizes
of DNA fragments obtained from the single, double and triple
digestions
4,363 bp
Figure 18.5
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18-37
Figure 19-22
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Figure 19-23
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