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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 18
RECOMBINANT DNA
TECHNOLOGY
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

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
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
To be continued
Figure 18.1

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
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
The Steps in Gene Cloning
Step 1. The chosen piece of DNA is ‘cut’ from the source organism using
restriction enzymes.
Step 2. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are
joined with the vector DNA by ligation.
Step 3. The vector is introduced into a host cell, often a bacterium or yeast, by
a process called transformation. The host cells copy the vector DNA along
with their own DNA, creating multiple copies of the inserted DNA.
Step 4. The vector DNA is isolated (or separated) from the host cells’ DNA and
purified.
DNA that has been ‘cut’ and ‘pasted’ from an organism into a vector is called
recombinant DNA. Because of this, DNA cloning is also called recombinant
DNA technology.

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
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
This step of the procedure
is termed transformation.
Cells that are able to take
up DNA are called
competent cells
Figure 18.2



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Nonrecombinant: recircularized
Recombinant: vector plus inserted cloned gene
Selection for vector: ampicillin resistance
Selection for recombinant vs. nonrecombinant
vector: b-galactosidase activity
Selection for gene of interest?

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

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
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
polyA tail
Figure 18.4

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)

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.

Polymerase Chain Reaction

Another way to copy DNA is a technique called
polymerase chain reaction (PCR)



It was developed by Kary Mullis in 1985
Unlike gene cloning, PCR can copy DNA without the
aid of vectors and host cells
The PCR method is outlined in Figure 18.6

The starting material for PCR includes

1. Template DNA


2. Oligonucleotide primers


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Provide the precursors for DNA synthesis
4. Taq polymerase


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Complementary to sequences at the ends of the DNA fragment
to be amplified
These are synthetic and about 15-20 nucleotides long
3. Deoxynucleoside triphosphates (dNTPs)


Contains the region that needs to be amplified
DNA polymerase isolated from the bacterium Thermus aquaticus
This thermostable enzyme is necessary because PCR involves
heating steps that inactivate most other DNA polymerases
Refer to Figure 18.6
The polymerase chain reaction (PCR)
Figure 18.6
Binding of the primers to
the DNA is called
annealing


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PCR is carried out in a thermocycler, which
automates the timing of each
cycle
All the ingredients are placed in
one tube
The experimenter sets the
machine to operate within a
defined temperature range and
number of cycles
Figure 18.6

The sequential process of
denaturing-annealingsynthesis is then repeated for
many cycles
With each successive cycle the relative amount of
this type of DNA fragment increases.
Therefore, after many cycles, the vast majority of
DNA fragments only contain the region that is
flanked by the two primers

A typical PCR run is likely to involve 20 to 30 cycles of replication



This takes a few hours to complete
After 20 cycles, a DNA sample will increase 220-fold (~ 1 million-fold)
After 30 cycles, a DNA sample will increase 230-fold (~ 1 billion-fold)
18.2 DETECTION OF GENES
AND GENE PRODUCTS

Molecular geneticists usually want to study particular
genes within the chromosomes of living species


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This presents a problem, because chromosomal DNA
contains thousands of different genes
The term gene detection refers to methods that distinguish
one particular gene from a mixture of thousands of genes
Scientists have also developed techniques to identify
gene products


RNA that is transcribed from a particular gene
Protein that is encoded in an mRNA