Chapter 19

DNA Technology

Lecture Presentations by
Nicole Tunbridge and
© 2021 Pearson Education Ltd. Kathleen Fitzpatrick
 Learning Objectives

At the end of this lecture, students should be able to:

• Describe the function of restriction enzymes and their use

in DNA technology
• Outline gene cloning
• Describe briefly polymerase chain reaction (PCR)
• Illustrate the process of gel electrophoresis
• Elucidate the applications of DNA technology

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Figure 19.1b

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Making Multiple Copies of a Gene or Other DNA
Segment
• To work directly with specific genes, scientists
prepare well-defined DNA segments in multiple
identical copies by a process called DNA cloning
• Plasmids are small, circular DNA molecules that
replicate separately from the bacterial chromosome
• Researchers can insert DNA into a plasmid to
produce a recombinant DNA molecule, which
contains DNA from two different sources

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 • Reproduction of a recombinant plasmid in a
bacterial cell results in cloning of the plasmid
including the foreign DNA
• This production of multiple copies of a single gene
is a type of DNA cloning called gene cloning
• A plasmid used to clone a foreign gene is called a
cloning vector
• Cloned genes are useful for making copies of a
particular gene and producing a protein product

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 Genetic Engineering/Biotechnology

• Methods for making recombinant DNA are

central to genetic engineering, the direct
manipulation of genes for practical purposes
• DNA technology has revolutionized
biotechnology, the manipulation of organisms
or their genetic components to make useful
products

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Gene cloning and Bacterium
Cell containing gene
of interest
its uses 1 Gene inserted into
plasmid

Bacterial Plasmid
chromosome
Gene of
Recombinant interest
DNA of
DNA (plasmid) chromosome
2 Plasmid put into
bacterial cell

Recombinant
bacterium

3 Host cell grown in culture

to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed

Interest by gene of interest
Copies of gene Protein harvested

4 Basic research and

Basic
various applications Basic
research research
on gene on protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
Using Restriction Enzymes to Make Recombinant
DNA
• Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called
restriction sites
• A restriction enzyme usually makes many cuts,
yielding restriction fragments

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• The most useful restriction enzymes cut DNA
in a staggered way, producing fragments
with “sticky ends” that bond with
complementary sticky ends of other fragments
• DNA ligase is an enzyme that seals the bonds
between restriction fragments

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Fig. 20-3-1
Restriction site

DNA 5¢ 3¢
3¢ 5¢

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end
Fig. 20-3-2
Restriction site

DNA 5¢ 3¢
3¢ 5¢

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

2 DNA fragment added

from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination

Fig. 20-3-3
Restriction site

DNA 5¢ 3¢
3¢ 5¢

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end

2 DNA fragment added

from another molecule
cut by same enzyme.
Base pairing occurs.

One possible combination

3 DNA ligase
seals strands.

Recombinant DNA molecule

Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR)
• The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
• A three-step cycle—heating, cooling, and
replication—brings about a chain reaction that
produces an exponentially growing population
of identical DNA molecules

• The key to PCR is an unusual, heat-stable

DNA polymerase called Taq polymerase

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 Fig. 20-8
5¢ 3¢
TECHNIQUE
Target
sequence

Genomic DNA 3¢ 5¢

Requirements:
1 Denaturation 5¢ 3¢
• dsDNA, o
(94-98 C)
• heat-resistant DNA pol
• all four nucleotides (in
3¢ 5¢
excess)
• two 15-20 nucleotide 2 Annealing

long ssDNA primers Cycle 1

(50-65 oC)
yields Primers
2
molecules

3 Extension

(70-80 oC)
New (5’à3’)
nucleo-
tides

Cycle 2
yields
4
molecules

Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
2n; after 30 cycles: ~1 billion sequence
copies
Fig. 20-8a

Requirements:
• dsDNA,
• heat-resistant DNA pol
• all four nucleotides (in excess)
• two 15-20 nucleotide long ssDNA primers

5¢ 3¢
TECHNIQUE
Target
sequence

Genomic DNA 3¢ 5¢

DNA pol from Thermus aquaticus (Taq polymerase). Functional up to 95 0C.

Fig. 20-8b

1 Denaturation 5¢ 3¢

(94-98 oC)

3¢ 5¢
2 Annealing

Cycle 1 (50-65 oC)

yields Primers
2
molecules

3 Extension

(70-80 oC)
New (5’à3’)
nucleo-
tides
Fig. 20-8c

Cycle 2
yields
4
molecules

2n; after 30 cycles: ~1 billion copies

Fig. 20-8d

Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence

• Within a few hours can make a billion copies of a segment even if it

originally made up less than 0.001% of total DNA in the sample
• PCR amplification occasionally incorporates errors into the
amplified strands and so cannot substitute for gene cloning in cells
Gel Electrophoresis

• One indirect method of rapidly analyzing and

comparing genomes is gel electrophoresis
• This technique uses a gel as a molecular sieve
to separate nucleic acids or proteins by size
• A current is applied that causes charged
molecules to move through the gel
• Molecules are sorted into “bands” by their size

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Figure 19.6

a buffer provide ions

to conduct electricity
Analyzing Gene Expression
• The most straightforward way to discover which
genes are expressed in certain cells is to identify
the mRNAs being made
• mRNA can be detected by nucleic acid
hybridization with complementary molecules
• These complementary molecules, of either DNA or
RNA, are nucleic acid probes

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 • In situ hybridization uses fluorescent dyes
attached to probes to identify the location of
specific mRNAs in place in the intact organism
• Different probes can be labeled with different
fluorescent dyes, sometimes with strikingly
beautiful results

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Figure 19.9

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Analyzing Gene Expression
• The most straightforward way to discover
which genes are expressed in certain cells is to
identify the mRNAs being made
• Reverse transcriptase-polymerase chain
reaction (RT-PCR) is useful for comparing
amounts of specific mRNAs in several samples at
the same time
• Reverse transcriptase is used to synthesize a
complementary DNA (cDNA) copy of each mRNA
in the sample
• A second DNA strand, complementary to the first is
synthesized by DNA polymerase

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Figure 19.10

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 • Next, PCR is used to amplify DNA segments of
interest from the cDNAs
• The products are run on a gel to determine which
samples expressed the gene of interest
• The presence, absence and relative expression of
mRNA can be compared

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 A. Gel electrophoresis B. Real-Time RT-PCR
Different gene expression levels Different gene expression levels
Applications of DNA technology: DNA Sequencing

• Relatively short DNA fragments can be

sequenced by the dideoxy chain termination
method
• Modified nucleotides called
dideoxyribonucleotides (ddNTP) attach to
synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct
fluorescent label that identifies the nucleotide
at the end of each DNA fragment
• The DNA sequence can be read from the
resulting spectrogram
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Fig. 20-12
TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides

(template strand) (fluorescently tagged)

dATP ddATP usually, 100 times

dCTP ddCTP lower concentration
dNTP DNA
dTTP ddTTP than deoxynucleotides
polymerase dGTP ddGTP

DNA (template Labeled strands

strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand ddNTP:
RESULTS Last base
they terminate DNA
of longest strand elongation
labeled because it lacks 3’-
strand
OH required for a
Last base phosphodiester bond
of shortest
labeled
strand
Application: Diagnosis of Diseases

• Scientists can diagnose many human genetic

disorders by using PCR and sequence-specific
primers, then sequencing the amplified product to
look for the disease-causing mutation
• Genetic disorders can also be tested for
using genetic markers that are linked to the
disease-causing allele

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Figure 19.15

SNPs (single nucleotide polymorphisms), single nucleotide

variants, are among the most useful genetic markers

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Editing Genes and Genomes
• The CRISPR-Cas9 system is a powerful new
technique for gene editing in living cells and
organisms
• It is an effective way for researchers to knock out a
given gene in order to study what the gene does
• Modifications of the technique allow researchers to
repair a gene that has a mutation (e.g., Gene
Therapy)

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Forensic Evidence and Genetic Profiles
• DNA testing can identify individuals with a high
degree of certainty in criminal and paternity cases
• An individual’s unique set of genetic markers, or
genetic profile, can be obtained by analysis of
tissue or body fluids
• Genetic profiles are currently analyzed using
genetic markers called short tandem repeats
(STRs)
• STRs are variations in the number of repeats of
specific DNA sequences; they are analyzed by
PCR and gel electrophoresis

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Figure 19.24

Values refer to
size of STR

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You should now be able to:

1. Describe the natural function of restriction enzymes and explain how

they are used in recombinant DNA technology
2. Outline the procedures for cloning a eukaryotic gene in a bacterial
plasmid
3. Describe the polymerase chain reaction (PCR) and explain the
advantages and limitations of this procedure
4. Explain how gel electrophoresis is used to analyze nucleic acids
5. Describe the application of DNA technology

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings