Recombinant DNA Technology

DNA technology allows us to study the

sequence, expression, and function of a gene

• DNA cloning allows researchers to

– Compare genes and alleles between
individuals
– Locate gene expression in a body
– Determine the role of a gene in an organism

• Several techniques are used to analyze the

DNA of genes

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 DNA cloning yields multiple copies of a gene
or other DNA segment
• To work directly with specific genes, scientists
prepare gene-sized pieces of DNA in identical
copies, a process called DNA cloning

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DNA Cloning and Its Applications: A Preview

• Most methods for cloning pieces of DNA in the

laboratory share general features, such as the
use of bacteria and their plasmids
• Plasmids are small circular DNA molecules
that replicate separately from the bacterial
chromosome
• Cloned genes are useful for making copies of a
particular gene and producing a protein product

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• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial
cell
• Reproduction in the bacterial cell results in
cloning of the plasmid including the foreign
DNA
• This results in the production of multiple copies
of a single gene

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 Cell containing gene
Bacterium
of interest
1 Gene inserted into
plasmid

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

Recombinant
bacterium
 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
• 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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 Restriction site

DNA 5 3
3 5

1 Restriction enzyme
cuts sugar-phosphate
backbones.

Sticky end
 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

 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

 Cloning a Eukaryotic Gene in a Bacterial
Plasmid

• In gene cloning, the original

plasmid is called a cloning vector
• A cloning vector is a DNA molecule
that can carry foreign DNA into a
host cell and replicate there

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 Producing Clones of Cells Carrying
Recombinant Plasmids
• Several steps are required to clone the
hummingbird β-globin gene in a bacterial
plasmid:
– The hummingbird genomic DNA and a
bacterial plasmid are isolated
– Both are digested with the same restriction
enzyme
– The fragments are mixed, and DNA ligase is
added to bond the fragment sticky ends

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 – Some recombinant plasmids now contain
hummingbird DNA
– The DNA mixture is added to bacteria that
have been genetically engineered to accept it
– The bacteria are plated on a type of agar that
selects for the bacteria with recombinant
plasmids
– This results in the cloning of many
hummingbird DNA fragments, including the β-
globin gene
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TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments
TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid
Recombinant plasmids
TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid
Recombinant plasmids

Bacteria carrying
plasmids
TECHNIQUE Hummingbird
cell
Bacterial cell
lacZ gene

Restriction Sticky Gene of interest

site ends
ampR gene Bacterial
Hummingbird
plasmid
DNA fragments

Nonrecombinant
plasmid
Recombinant plasmids

Bacteria carrying
plasmids

RESULTS

Colony carrying non- Colony carrying recombinant

recombinant plasmid plasmid with disrupted lacZ gene
with intact lacZ gene

One of many
bacterial
clones
 Storing Cloned Genes in DNA Libraries

• A genomic library that is made using bacteria

is the collection of recombinant vector clones
produced by cloning DNA fragments from an
entire genome
• A genomic library that is made using
bacteriophages is stored as a collection of
phage clones

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 Foreign genome
cut up with
restriction
enzyme
or

Recombinant
phage DNA
Bacterial Recombinant
clones plasmids Phage
clones

(a) Plasmid library (b) Phage library

 Screening a Library for Clones Carrying a
Gene of Interest
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid
hybridization

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• A probe can be synthesized that is
complementary to the gene of interest
• For example, if the desired gene is

5 … G G C T A A C T T A G C … 3

– Then we would synthesize this probe

3 C C G A T T G A A T C G 5

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• The DNA probe can be used to screen
a large number of clones
simultaneously for the gene of interest
• Once identified, the clone carrying the
gene of interest can be cultured

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TECHNIQUE

Radioactively
labeled probe
molecules Probe
DNA Gene of
Multiwell plates interest
holding library Single-stranded Film
clones DNA from cell

Nylon membrane
Nylon
Location of
membrane
DNA with the
complementary
sequence
 Introducing recombinant DNA into eukaryotic cells

• One method of introducing recombinant DNA

into eukaryotic cells is electroporation,
applying a brief electrical pulse to create
temporary holes in plasma membranes
• Alternatively, scientists can inject DNA into
cells using microscopically thin needles
• Once inside the cell, the DNA is incorporated
into the cell’s DNA by natural genetic
recombination

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 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

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Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
 The PCR method
• The basic steps of PCR are as follows:
1. The reaction mixture is heated to separate the strands
of the DNA double helices.
2. The strands are cooled. As they cool, primer
molecules hydrogen-bond to their target sequences
on the DNA.
3. A heat-stable DNA polymerase builds new DNA
strands by extending the primers in the
5→3 direction.
• These three steps are repeated over and over,
doubling the amount of DNA after each three-step
cycle.

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 5 3
TECHNIQUE
Target
sequence

Genomic DNA 3 5

1 Denaturation 5 3

3 5

2 Annealing

Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleo-
tides

Cycle 2
yields
4
molecules

Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
 Gel Electrophoresis and Southern Blotting

• 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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 Gel electrophoresis can be used to separate DNA
molecules based on size as follows:

1. A DNA sample is placed at one end of a porous gel.

2. Current is applied, and DNA molecules move from the
negative electrode toward the positive electrode.
3. Shorter DNA fragments move through the gel matrix
more quickly and travel farther through the gel.
4. DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence.
5. Each band is a collection of DNA molecules of the
same length.

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 TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules
RESULTS
 RFLPs can be used to detect differences in
DNA sequences

• Geneticists have cataloged many single-base-pair

variations in the genome.
• Such a variation found in at least 1% of the
population is called a single nucleotide
polymorphism (SNP, pronounced ―snip‖).
• SNPs occur on average about once in 100 to 300
base pairs in the human genome,
• in the coding sequences of genes and
• in noncoding sequences between genes.

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 RFLPs can be used to detect differences in
DNA sequences

• SNPs may alter a restriction site—the sequence

recognized by a restriction enzyme.
• Such alterations change the lengths of the
restriction fragments formed by that enzyme when it
cuts the DNA.
• A sequence variation of this type is called a
restriction fragment length polymorphism
(RFLP, pronounced ―rif-lip‖).
• Thus, RFLPs can serve as genetic markers for
particular loci in the genome.

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• In restriction fragment analysis, DNA
fragments produced by restriction enzyme
digestion of a DNA molecule are sorted by gel
electrophoresis
• Restriction fragment analysis is useful for
comparing two different DNA molecules, such
as two alleles for a gene
• The procedure is also used to prepare pure
samples of individual fragments

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 Normal -globin allele Normal Sickle-cell
allele allele

175 bp 201 bp Large fragment

DdeI DdeI DdeI DdeI Large

fragment
Sickle-cell mutant -globin allele

376 bp
376 bp Large fragment 201 bp
175 bp
DdeI DdeI DdeI

(a) DdeI restriction sites in normal and (b) Electrophoresis of restriction fragments
sickle-cell alleles of -globin gene from normal and sickle-cell alleles
• A technique called Southern blotting
combines gel electrophoresis of DNA
fragments with nucleic acid
hybridization
• Specific DNA fragments can be
identified by Southern blotting, using
labeled probes that hybridize to the
DNA immobilized on a ―blot‖ of gel
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 TECHNIQUE
Heavy
Restriction I II III weight
DNA + restriction enzyme fragments Nitrocellulose
membrane (blot)

Gel

Sponge

I Normal II Sickle-cell III Heterozygote Paper

-globin allele Alkaline
towels
allele solution

1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled
probe for -globin gene

Probe base-pairs
I II III with fragments I II III

Fragment from
sickle-cell
-globin allele Film
over
Fragment from blot
normal -globin
Nitrocellulose blot allele
4 Hybridization with radioactive probe 5 Probe detection
 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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TECHNIQUE

DNA Primer Deoxyribonucleotides Dideoxyribonucleotides

(template strand) (fluorescently tagged)

dATP ddATP
dCTP ddCTP

DNA dTTP ddTTP

polymerase dGTP ddGTP
TECHNIQUE

DNA (template Labeled strands

strand)

Shortest Longest

Direction
of movement Longest labeled strand
of strands

Detector

Laser
Shortest labeled strand
RESULTS Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
 Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or

genetic profile, can be obtained by analysis of
tissue or body fluids
• Genetic profiles can be used to provide
evidence in criminal and paternity cases and to
identify human remains

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• Even more sensitive is the use of genetic
markers called short tandem repeats (STRs),
which are variations in the number of repeats
of specific DNA sequences
• PCR and gel electrophoresis are used to
amplify and then identify STRs of different
lengths
• The probability that two people who are not
identical twins have the same STR markers is
exceptionally small

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 STR site 1 STR site 2
AGAT GATA
Crime scene
DNA

The number of short tandem The number of short tandem

repeats match repeats do not match

Suspect’s
DNA
AGAT GATA

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(a) This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.

Source of STR STR STR

sample marker 1 marker 2 marker 3

Semen on victim 17, 19 13, 16 12, 12

Earl Washington 16, 18 14, 15 11, 12

Kenneth Tinsley 17, 19 13, 16 12, 12

(b) These and other STR data exonerated Washington and

led Tinsley to plead guilty to the murder.
 Genomics is the scientific study of whole
genomes

• Genomics is the study of an organism’s complete

set of genes and their interactions.
• Initial studies focused on prokaryotic genomes.
• Many eukaryotic genomes have since been
investigated.
• As of 2013, the genomes of nearly 7,000 species
have been completed, and thousands more are in
progress.

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 The Human Genome Project

• The goals of the Human Genome Project (HGP)

included
• determining the nucleotide sequence of all DNA in
the human genome and
• identifying the location and sequence of every
human gene.

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 • Results of the Human Genome Project indicate
that
• humans have about 21,000 genes in 3 billion
nucleotide pairs,
• only 1.5% of the DNA codes for proteins, tRNAs, or
rRNAs, and
• the remaining 98.5% of the DNA is noncoding DNA.

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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

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50 µm
 Studying the Expression of Interacting
Groups of Genes

• Automation has allowed scientists to

measure expression of thousands of
genes at one time using DNA
microarray assays
• DNA microarray assays compare
patterns of gene expression in different
tissues, at different times, or under
different conditions
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TECHNIQUE
Tissue sample
1 Isolate mRNA.

2 Make cDNA by reverse mRNA molecules

transcription, using
fluorescently labeled
nucleotides.

Labeled cDNA molecules

(single strands)
3 Apply the cDNA mixture to a DNA fragments
microarray, a different gene in representing
each spot. The cDNA hybridizes specific genes
with any complementary DNA on
the microarray.
DNA microarray

DNA microarray
4 Rinse off excess cDNA; scan
with 2,400
microarray for fluorescence.
human genes
Each fluorescent spot represents a
gene expressed in the tissue sample.
 Important Areas of DNA Technology
Big deals

Gene Cloning Genetically Modified

Organisms

DNA Profiling Genomics

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 DNA technologies affect our lives in many other ways.

• Gene cloning is used to produce medical and

industrial products.
• DNA profiling has changed the field of forensic
science.
• New technologies produce valuable data for biological
research.
• DNA can be used to investigate historical questions.

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