Overview

 Introduction to Recombinant DNA Technology and DNA

Cloning
 What Makes a Good Vector?
 How Do You Identify and Clone a Gene of Interest?
 Laboratory Techniques and Applications of Recombinant
DNA Technology

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 Introduction to Recombinant DNA Technology and DNA
Cloning

 1970s gene cloning became a reality

◦ Clone – a molecule, cell, or organism that was produced
from another single entity
 Gene cloning: generate multiple copies of a piece of DNA in a
form that can be used (e.g., expressed in a bacterium or yeast that
can synthesize a protein)
 Made possible by the discovery of
◦ Restriction Enzymes – DNA cutting enzymes
◦ Plasmid DNA Vectors – circular form of
self-replicating DNA
 Can be manipulated to carry other pieces of DNA
 Can be introduced into a suitable host to get many copies of
the cloned piece of DNA

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 Traditional cloning strategy

Goal: insert a particular

piece of DNA into a cloning
vector

 Purify the donor DNA and

vector DNA
 Cleave the donor and
vector DNA with a
restriction enzyme
 Join the donor DNA with
vector DNA
 Identify and isolate the
desired recombinant clones
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 Restriction Enzymes
(Endonucleases that cleave DNA at specific sites)
◦ Primarily found in bacteria
◦ Part of the restriction-modification system
 Modify self and restrict non-self
◦ Cut DNA by cleaving the phosphodiester bond that joins adjacent
nucleotides in a DNA strand
◦ Bind to, recognize, and cut DNA within specific sequences of
bases called a recognition sequence or restriction site
 Recognition sequences are palindromes
 Cohesive (sticky) ends – overhanging single-stranded ends
 Blunt ends – double-stranded, non-overhanging ends
 There are 4 or 6 bp cutters because they recognize restriction sites
with a sequence of 4 or 6 nucleotides

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 Restriction –Modification system

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 Restriction-Modification system

Restriction-modification (R-M)
systems as defense
mechanisms. R-M systems
recognize the methylation
status of incoming foreign
DNA, e.g., phage genomes.
restriction methyltransferase Methylated sequences are
endonucleas recognized as self, while
e recognition sequences on the
incoming DNA lacking
methylation are recognized as
nonself and are cleaved by
the restriction endonuclease
(REase). The methylation
status at the genomic
recognition sites is maintained
by the cognate
methyltransferase (MTase) of
the R-M system.

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 Vectors – Plasmids
 Plasmids are naturally occurring extra- Ampr
chromosomal DNA molecules.
Ori
pBR322
 Plasmids are circular, double-stranded 4361bp

DNA.
Tetr
 Plasmids are the means by which
antibiotic resistance is often transferred
from one bacteria to another.
LacZ
 Plasmids can be cleaved by restriction MCS

enzymes, leaving sticky or blunt ends.

pUC18
 Artificial plasmids can be constructed by Ori

linking new DNA fragments to the sticky Ampr

ends of plasmid.

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 Cloning Vectors

Older cloning vector

 A cloning vector is a plasmid that can be modified to
carry new genes. Ampr

 Plasmids useful as cloning vectors must have: Ori

pBR322
◦ An origin of replication. 4361bp

◦ A selectable marker (antibiotic resistance gene, such Tetr

as ampr and tetr).
◦ Multiple cloning site (MCS) (site where insertion of
foreign DNA will not disrupt replication or inactivate Newer cloning vector
essential markers).
LacZ
◦ Lac Z gene allows for blue-white selection MCS

 If foreign insert present, Lac Z gene’s ORF is disrupted.

pUC18
 In the absence of the foreign insert Lac Z gene is intact Ori

Ampr

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 DNA Cloning

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 Transformation of Bacterial Cells
Once the plasmid contains donor DNA, we need to get that DNA into a recipient cell in
which it can replicate.

Recall that transformation is a

Calcium chloride method
naturally occurring process in
treat bacterial cells with calcium chloride
bacteria.
add plasmid DNA to cells chilled on ice
heat the cell and DNA mixture
plasmid DNA enters bacterial cells and is
replicated and expressed

electroporation (more modern)

apply a voltage across the membrane to
induce the formation of temporary
pores in the bilayer
rapid and efficient
can be used to introduce DNA into
multiple cell types

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 Selection of Recombinant Bacteria

◦ Selection is a process designed to facilitate the

identification of recombinant bacteria while preventing
the growth of non-transformed bacteria
 Antibiotic selection
◦ Transformed bacteria, carrying naïve vs. chimeric
plasmids can be differentiated by their blue color, based
on the presence or absence of an intact Lac Z gene
 Blue-white selection

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 DNA Cloning Process

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 Gene libraries
 Creating DNA Libraries
◦ Collections of cloned DNA fragments from a particular organism
contained within bacteria or viruses as the host

◦ Screened to pick out

different genes of interest

 Two Types of Libraries

◦ Genomic DNA libraries
◦ Complementary DNA libraries (cDNA libraries)

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 Genomic Libraries
◦ Chromosomal DNA from the tissue of interest is isolated
and digested with restriction enzyme
◦ Vector is digested with same enzyme and DNA ligase is
used to ligate genomic DNA fragments and vector DNA
◦ Recombinant vectors are used to transform bacteria
◦ Disadvantages
 Non-protein coding pieces of DNA (introns) are cloned in
addition to exons; majority of genomic DNA is introns in
eukaryotes so majority of the library will contain non-coding
pieces of DNA
 Many organisms have very large genome, so searching for
gene of interest is difficult at best

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 cDNA Libraries
◦ mRNA from tissue of interest is isolated
◦ Converted to a double-stranded DNA by
using the enzyme reverse transcriptase
 Called complementary DNA (cDNA)
because it is an exact copy of the mRNA
◦ mRNA is degraded
◦ DNA polymerase used to create the second
strand of DNA
◦ Short linker sequences are added to the
end of the cDNA
 Contain restriction enzyme recognition
sites
◦ Cut with restriction enzyme, cut vector with
same enzyme, ligate fragments to create
recombinant vectors
◦ Vectors used to transform bacteria

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 cDNA Libraries Contd..
◦ Advantage
 Collection of actively expressed genes in the cells or tissues
from which the mRNA was isolated
 Introns are NOT cloned
 Can be created and screened to isolate genes that are
primarily expressed only under certain conditions in a tissue
◦ Disadvantage
 Can be difficult to make the cDNA library if a source tissue
with an abundant amount of mRNA for the gene is not
available

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 Genomic vs cDNA Libraries

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 How Do You Identify and Clone a Gene of
Interest?
 Libraries are “screened” to identify the gene of interest
 Colony hybridization
◦ Bacterial colonies containing recombinant DNA are grown on an
agar plate
◦ Nylon or nitrocellulose filter is placed over the plate and some of
the bacterial colonies stick to the filter at the exact location they
were on the plate
◦ Treat filter with alkaline solution to lyse the cells and denature the
DNA
◦ Denatured DNA binds to filter as single-stranded DNA
◦ Filter is incubated with a probe
 DNA fragment that is complementary to the gene of interest
 Labeled with P32 or Fluorescent tag for easy detection
◦ Probe binds by hydrogen bonding to complementary sequences
on the filter

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 Colony Hybridization
◦ Filter is washed to remove excess unbound probe
◦ Filter is exposed to film – autoradiography
 Anywhere probe has bound, light will be emitted and expose
silver grains in the film
 Film is developed to create a permanent record of the colony
hybridization
◦ Film is then compared to the original agar plate to
identify which colonies contained recombinant plasmid
with the gene of interest

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 Colony Hybridization

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 Colony Hybridization Contd…
 Colony Hybridization
◦ Type of probe used depends on what is already known about
the gene of interest
◦ Example: use mouse or rat probe to screen a human library
because many genes between these species are similar
◦ If gene sequence has NOT been cloned in another species
but something is known about the protein, what can be
done?
 Rarely results in the cloning of the full-length gene
◦ Usually get small pieces of the gene; the pieces are
sequenced and scientists look for overlapping sequences
◦ Look for start and stop codons to know when the full length of
the gene is obtained

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 Newer Approach: PCR cloning

• DNA fragment of interest can be amplified and inserted

into a plasmid or other vector.
Advantages over traditional cloning:
- rapid
- can clone DNA fragments not available in large amounts
• Handicap – one needs to know the sequence of the
DNA flanking the gene of inerest.
• Primers designed to anneal to regions flanking
gene of interest.
• Primers can also contain sequences with restriction
sites so amplified gene can easily be inserted into
a plasmid.
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 Polymerase Chain Reaction

◦ Developed in the 1980s by Kary Mullis

◦ Technique for making copies, or amplifying, a specific
sequence of DNA in a short period of time
◦ Process
 Target DNA to be amplified is added to a tube, mixed with
nucleotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA
polymerase.
 Primers are added – short single-stranded DNA
oligonucleotides (20–30bp long)
 Reaction tube is placed in an instrument called a
thermocycler

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 Process

◦ Thermocycler will take DNA through a series of reactions

called a PCR cycle
◦ Each cycle consists of three stages
 Denaturation (94-960C)
 Annealing (hybridization) (50 -650C)
 Extension (elongation) (70 -750C)
◦ At the end of one cycle, the amount of DNA has doubled
◦ Cycles are repeated 20–30 times

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 The first round of PCR

94°C 37-65°C 70-75°C

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 PCR increases the yield of DNA exponentially

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 Polymerase Chain Reaction (PCR)
A thermophilic (heat-loving) bacteria called Thermus aquaticus is
the source of Taq DNA polymerase used in PCR reactions.

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 PCR contd…
 The type of DNA polymerase used is very important
◦ Taq DNA polymerase – isolated from a species known as Thermus
aquaticus that thrives in hot springs (1st isolated from Yellowstone
National Park)
 Advantage of PCR
◦ Ability to amplify millions of copies of target DNA from a very small
amount of starting material in a short period of time
 Applications
◦ Making DNA probes
◦ Studying gene expression (quantitative real-time PCR)
◦ Detection of viral and bacterial infections
◦ Diagnosis of genetic conditions
◦ Detection of trace amounts of DNA from tissue found at crime scene

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 Cloning PCR Products
◦ Is rapid and effective
◦ Disadvantage
 Need to know something about the DNA sequence that
flanks the gene of interest to design primers
 Assume the human genome project was not completed but you
wanted to clone growth hormone from humans, what sequence
would you use to design PCR primers?
◦ Advantages
 Includes restriction enzyme recognition sequences in the
primers
 Uses T vector
 Taq polymerase puts a single adenine nucleotide on the 3’ end
of all PCR products

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 Subcloning PCR products

then what?
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 What can you do with a cloned gene?
applications of recombinant DNA technology

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 Recombinant DNA products

If we want to use a
bacterial expression
host to produce an
animal protein,
should we simply
amplify genomic
DNA from the source
organism and then
stick it in bacteria?

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 Types of Vectors
◦ Bacterial plasmid vectors
◦ Bacteriophage vectors
◦ Cosmid vectors
◦ Expression vectors
◦ Bacterial Artificial Chromosomes (BAC)
◦ Yeast Artificial Chromosomes (YAC)
◦ Shuttle vectors
◦ Ti vectors

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