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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 18 Part 2
RECOMBINANT
DNA TECHNOLOGY
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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
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18-38

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
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18-39
The polymerase chain reaction (PCR)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 18.6
Binding of the primers to the
DNA is called annealing



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
18-40
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)
18-41
18.2 DETECTION OF GENES
AND GENE PRODUCTS

Molecular geneticists usually want to study particular
genes within the chromosomes of living species



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
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18-44
DNA Libraries

A DNA library is a collection of thousands of cloned
fragments of DNA

When the starting material is chromosomal DNA, the
library is called a genomic library

A cDNA library contains hybrid vectors with cDNA inserts
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18-45
DNA Libraries
• A genomic library
– Contains fragments of chromosomal DNA
– Includes gene exons/introns and nongene
sequences
• A complementary DNA (cDNA) library
– Is made by cloning DNA made in vitro by
reverse transcription of all the mRNA produced
by a particular cell
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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
18-15
Cleave DNA
with restriction
enzyme
Figure 18.7
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Figure 18.7
18-47

In most cloning experiments, the ultimate goal is to
clone a specific gene

For example, suppose that a geneticist wishes to
clone the rat b-globin gene



Only a small percentage of the hybrid vectors in a DNA
library would actually contain the gene
Therefore, geneticists must have a way to distinguish
those rare colonies from all the others
This can be accomplished by using a DNA probe in
a procedure called colony hybridization

Refer to Figure 18.8
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18-48
The filter is treated with detergent (SDS) to permealize the bacteria
NaOH is added to denature the DNA
A radioactively labeled probe that is complementary to the b-globin
gene is then added
Figure 18.8
18-49
Screening DNA Libraries
• screening a DNA library for a particular
nucleotide sequence
– Fragment of a gene
– Related gene
• screening a DNA library based on detection of
protein expression from cloned gene
– Antibody probes
– Protein probes
– DNA binding sites (transcription factors)
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Screening DNA Libraries

What if a scientist is looking for a novel type of
gene that no one else has ever cloned from any
species?


If the protein of interest has been previously isolated,
amino acid sequences are obtained from it
The researcher can use these amino sequences to
design short DNA probes that can bind to the protein’s
coding sequence
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18-50
Southern Blotting

Southern blotting can detect the presence of a
particular gene sequence within a mixture of many


It was developed by E. M. Southern in 1975
Southern blotting has several uses





1. determine copy number of a gene in a genome
2. detect variations in gene structure
3. identify gene families
4. identify homologous genes among different species
5. characterize structure of cloned genes
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18-51

Prior to a Southern blotting experiment, the gene of
interest, or a fragment of a gene, has been cloned

This cloned DNA is labeled (e.g., radiolabeled) and used
as a probe

The probe will be able to detect the gene of interest
within a mixture of many DNA fragments
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18-52
a) The steps in
Southern blotting
b) The transfer step
An alternative type
of transfer uses a
vaccuum
or nylon
Figure 18.9
18-53
a) The steps in
Southern blotting
A common labeling method is
the use of the radioisotope 32P
Conditions of high temperature
or high salt concentrations
Probe DNA and chromosomal
fragment must be nearly
identical to hybridize
The filter is placed in a solution containing a labeled probe
The binding can be done under conditions of low or high stringency
Excess probe is washed away and the filter is exposed to X-ray film
Temperature and/or ionic
strength are lower
Probe DNA and chromosomal
fragment must be similar but not
necessarily identical to hybridize
Gene of interest is
found only in single
copy in the genome
Figure 18.9
Gene is member of a gene
family composed of three
distinct members
18-54
Northern Blotting

Northern blotting is used to identify a specific RNA
within a mixture of many RNA molecules


It was not named after anyone called Northern!
Northern blotting has several uses

1. It can determine if a specific gene is transcribed in a
particular cell type


2. It can determine if a specific gene is transcribed at a
particular stage of development


Nerve vs. muscle cells
Fetal vs. adult cells
3. It can reveal if a pre-mRNA is alternatively spliced
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18-55


Northern blotting is similar to Southern blotting
It is carried out in the following manner


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RNA is extracted from the cell(s) and purified
It is separated by gel electrophoresis
It is then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a
radioactive DNA probe
The filters are then exposed to an X-ray film


RNAs that are complementary to the radiolabeled probe are
detected as dark bands on the X-ray film
Figure 18.10 shows the results of a Northern blot for
mRNA encoding a protein called tropomyosin
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18-56
Figure 18.10

Smooth and striated muscles produce a larger amount of
tropomyosin mRNA than do brain cells


This is expected because tropomyosin plays a role in muscle
contraction
The three mRNAs have different molecular weights

This indicates that the pre-mRNA is alternatively spliced
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18-57
Western Blotting

Western blotting is used to identify a specific
protein within a mixture of many protein molecules


Again, it was not named after anyone called Western!
Western blotting has several uses

1. It can determine if a specific protein is made in a
particular cell type


Red blood cells vs. brain cells
2. It can determine if a specific protein is made at a
particular stage of development

Fetal vs. adult cells
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18-58

Western blotting is carried out as such


Proteins are extracted from the cell(s) and purified
They are then separated by SDS-PAGE

They are first dissolved in the detergent sodium dodecyl sulfate
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The secondary antibody is also conjugated to alkaline phosphatase
The colorless dye XP is added


The negatively charged proteins are then separated by
polyacrylamide gel electrophoresis
They are then blotted onto nitrocellulose or nylon filters
The filters are placed into a solution containing a primary
antibody (recognizes the protein of interest)
A secondary antibody, which recognizes the constant
region of the primary antibody, is then added


This denatures proteins and coats them with negative charges
Alkaline phosphatase converts the dye to a black compound
Thus proteins of interest are indicated by dark bands
18-59


Figure 18.11 shows the results of a Western blot for
the b-globin polypeptide
This experiment indicates that b-globin is made in
red blood cells but not in brain or intestinal cells
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18-60
18.3 ANALYSIS & ALTERATION OF
DNA SEQUENCES

Analyzing and altering DNA sequences is a powerful
approach to understanding genetics

A technique called DNA sequencing enables researchers to
determine the base sequence of DNA


It is one of the most important tools for exploring genetics at the
molecular level
Another technique known as site-directed mutagenesis
allows scientists to change the sequence of DNA

This too provides information regarding the function of genes
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18-66
DNA Sequencing

During the 1970s two DNA sequencing methods
were devised



One method, developed by Alan Maxam and Walter
Gilbert, involves the base-specific cleavage of DNA
The other method, developed by Frederick Sanger, is
known as dideoxy sequencing
The dideoxy method has become the more popular
and will therefore be discussed here
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18-67
DNA Sequencing


DNA polymerase connects adjacent deoxynucleotides by covalently
linking the 5’–P of one and the 3’–OH of the other (Refer to Fig. 11.10)
Nucleotides missing that 3’–OH can be synthesized
Figure 18.14

2’, 3’-dideoxyadenosine triphosphate
if a dideoxynucleotide is added to a growing DNA strand,
the strand can no longer grow: chain termination
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18-68

Prior to DNA sequencing, the DNA to be sequenced must be
obtained in large amounts

This is accomplished using cloning or PCR techniques

In many sequencing experiments, the target DNA is cloned
into the vector at a site adjacent to a primer annealing site

If double-stranded DNA is used as the template, it must be
denatured at the beginning of the experiment
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18-69
Figure 18.15
The newly-made DNA fragments can be
separated according to their length by
running them on an acrylamide gel
They can then be visualized as bands
when the gel is exposed to X-ray film
Many copies of the primer, template DNA and
radiolabeled dNTPs are mixed together
They are then divided into four tubes, each containing
a low concentration of a different dideoxynucleotide
Sequencing
ladder
18-70

An important innovation in the
method of dideoxy sequencing
is automated sequencing

It uses a single tube containing all
four dideoxyribonucleotides

However, each type (ddA, ddT,
ddG, and ddC) has a differentcolored fluorescent label attached

After incubation and
polymerization, the sample is
loaded into a single lane of a gel
Figure 18.16
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18-71



The procedure is automated using a laser and fluorescent
detector
The fragments are separated by gel electrophoresis
As each band comes off the bottom of the gel, the fluorescent
dye is excited by the laser

The fluorescence emission is recorded by the fluorescence detector

The detector reads the level of fluorescence at four wavelengths
Figure 18.16
18-72