Asst. Prof. Dr.

Sawitree Chiampanichayakul
Dept. of Medical Technology
Fac. of Associated Medical Sciences
Chiang Mai University
DNA Cloning – the act of making many identical copies
of a particular piece of DNA (often a gene).
 DNA Cloning

There are several steps involved in cloning a gene in a cell. The

specific steps in an individual procedure may vary, but most follow
these steps:

Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
 Overall of DNA Cloning
Isolation of
DNA

Selection of cloning
vector act as a vehicle

Ligating the DNA into a

vector to make
recombinant DNA

Introduction of Selection of host

recombinant DNA cells containing
into host cells the recombinant
DNA
 How is the DNA removed from the cells?
 How is the DNA cut into pieces?
Purification of DNA from living cells

Total cell DNA (DNA of interest)

Genomic DNA library
cDNA library

Vector DNA
 Plasmid DNA
 Phage DNA
 Genomic DNA Libraries
Genomic library is all DNA, introns, exons and non-coding

The genomic DNA is digested by a restriction endonuclease,

All fragments cloned at random into a plasmid vector.

Cultures of the bacteria, with each containing only a fraction of

the genome,

Collectively contain all the genes and are called a library.

Construction of a human
genomic DNA library
Genomic DNA Libraries

Foreign genome
cut up with
restriction
enzyme

or

Bacterial Recombinant
plasmids Recombinant
clones phage DNA Phage
clones

(a) Plasmid library (b) Phage library

 cDNA library

 cDNA made in vitro by reverse transcription of all the

mRNA produced by a particular cell.
Making cDNA from
mRNA using reverse
transcriptase and
DNA polymerase
• Plasmid DNA : Stringent plasmid
: Relaxed plasmid
Plasmid DNA separation
Plasmid DNA separation on the basis of size
Physical property: most plasmid DNA are much smaller than
bacterial DNA
Treatment cells with EDTA and lysozyme in the presence of
sucrose, the spheroplasts are formed
Cell lysis is induced by non-ionic detergent (Triton X-100)
After centrifugation, the cleared lysate consisting of plasmid
DNA
Plasmid DNA separation
Plasmid DNA separation on the basis of conformation
Most plasmid DNA exist in the cell as supercoiled molecules
Two different methods are commonly used:
Alkaline denaturation
Ethidium bromide-Caesium chloride density gradient
centrifugation
Alkaline denaturation

Linear DNA

pH 12-12.5

Supercoiled Single-stranded
plasmids linear DNA

pH 7

Tangled mass of
linear DNA
Ethidium bromide-Caesium chloride density gradient centrifugation

CsCl CsCl+EtBr

Protein Protein
1.6
1.65
1.7 DNA Linear DNA
1.75
Supercolied DNA
1.8
RNA RNA
How are the pieces of DNA put back together?
Restriction enzyme

 Linn S. and Arber, W. (1960s) found RE in bacteria, where

they are used as a defense against bacteriophage infection by
cutting bacteriophage DNA inside of bacteria.
 Bacterial DNA is protected from restriction enzymes by the
addition of methyl groups to bacterial DNA to adenine or
cytosine.
 Also called “restriction endonucleases”, they cut DNA like
scissors at specific sites called “restriction sites” or
“restriction sequences”.
 They cut across the sugar-phosphate backbone of DNA by
breaking the covalent bond holding the sugar and phosphate
together.
 Sticky end

Sticky end
 Restriction enzyme Type II

Restriction sequences are usually four to six base pairs in

length, and are palindromic, which means that the sequence of
both DNA strands are the same when the top strand is read
from left to right, and then when the bottom strand is read from
right to left.
Can cut in to result in pieces of DNA with two possible ends:
Blunt ends: The enzyme cuts directly across the two
strands of DNA.
Sticky ends: The enzyme cuts both strands in different
places, leaving a short single-stranded piece of DNA to hang
over the end of the molecule. This piece can base
pair with other single-stranded pieces of DNA to form
recombinant DNA
 Separating Restriction Fragments and
Visualizing DNA

 Pieces of DNA are generated by restriction enzymes

can be separated and viewed.
Restriction enzymes and recombinant DNA
Restriction site

DNA 5′ GAATTC 3′
3′ CTTAAG
5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow

G AATTC

CTTAA G

Sticky end
2 DNA fragment from
another source is added. AATTC
G
Base pairing of sticky G
CTTAA
ends produces various
combinations. Fragment from different
DNA molecule cut by the
same restriction enzyme

G AATT C G AATTC
C TTAA G CTTAA G

3 DNA ligase One possible combination

seals the strands.

Recombinant DNA molecule

 DNA Cloning

There are several steps involved in cloning a gene in a cell. The

specific steps in an individual procedure may vary, but most follow
these steps:

Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Cloning Vectors

 A vector is used to amplify a single molecule of DNA

into many copes.
 A DNA fragment must be inserted into a cloning
vector.
 A cloning vector is a DNA molecule that has an origin
of replication and is capable of replicating in a
bacterial cell.
 Most vectors are genetically engineered plasmids or
phages.
 Cloning Vectors
The DNA of interest is inserted into a cloning vector to transport it
into the host cell.
The vector needs to have the following characteristics :

 Have an origin of replication so that the DNA can be replicated

within a host cell.
 Be small enough to be isolated without being degraded during
purification.
 Have several unique restriction sites for cloning a DNA fragment
(called a “multiple cloning site,” or “MCS”) so that the vector
will be cut only once to open it.
 Have selectable markers for determining whether the cloning
vehicle has been transferred into cells and to indicate whether
the foreign DNA has been inserted into the vector.
Cloning Vectors
A. Bacterial Vectors
Plasmids
Bacteriophage
Cosmids
A. Vectors for Other Organisms
Yeast Artificial Chromosomes (YACs)
Bacterial Artificial Chromosomes (BACs)
Plant Cloning Vectors
Mammalian Cell Vectors
 Bacterial Vectors
Plasmid
Extrachromosomal DNA in a bacterial cell which can replicate
independently.
Drug resistance plasmids are not essential for the cell's growth,
but confer antibiotic resistance.
Plasmids used for molecular cloning have been artificially
created by recombining fragments of various existing plasmids.
Plasmids contain multiple cloning sites with several restriction
endonuclease sites.
Engineered as vectors that fit pieces of DNA of up to 10
kilobases in length
A bacterial plasmid used
as a cloning vector
Plasmid Cloning Vectors
 Plasmids are circular, double-stranded DNA
molecules that exist in bacteria and in the nuclei
of some eukaryotic cells.
 They can replicate independently of the host cell.
The size of plasmids ranges from a few kb to
near 100 kb
 Can hold up to 10 kb fragments
 Plasmids have an origin of replication, antibiotic
resistance genes as markers, and several unique
restriction sites.
 After culture growth, the clone fragment can be
recovered easily. The cells are lysed and the
DNA is isolated and purified.
 A DNA fragment can be kept indefinitely if
mixed with glycerol in a –70oC freezer.
 pBR322 :
• The molecule is small, and can be isolated easily.
• This vector can accommodate DNA of up to 5 to 10 kb.
• pBR322 has several unique restriction sites where the plasmid
can be opened for inserting a DNA fragment.
• The genes encoding resistance to ampicillin (ampr) and
tetracycline (tetr) are used for plasmid and DNA insert selection.
• Provides for the insertable selection of a selectable marker:
• If one of the antibiotic resistance genes is broken with the
DNA of interest, then the bacteria receiving the plasmid will be
sensitive to the antibiotic and die if treated with the antibiotic.
• A method that determines if a recombinant plasmid was
created correctly and inserted correctly into the bacteria.
pUC19

Polylinker:
restriction
sites
lacZ+
gene

Origin
Ampicillin sequence
resistance
gene
Fig. 7.5
*Cut with same
restriction enzyme
*DNA ligase
Bacteriophage cloning vector

A virus that infects bacteria, and whose DNA can be engineered

into a cloning vector.
Kills bacteria by two pathways:
Lytic pathway: Bacteriophage DNA is inserted into the
bacteria, and phage DNA and phage proteins are made,
assembled, and burst out of the bacteria, causing the bacteria
to burst (called “lysis”).
Lysogenic pathway: The bacteriophage genome is
integrated into the bacterial DNA, replicating along with the
bacterial cell genome.
Phage Cloning Vectors
 Fragments up to 23 kb can be may be accommodated by a phage
vector
 Lambda phage and M13 phage are most common phage
 Segments of the Lambda DNA is removed and a stuffer fragment
is put in.
 The stuffer fragment keeps the vector at a correct size and carries
marker genes that are removed when foreign DNA is inserted into
the vector.
 Example: Charon 4A Lambda
 Cosmid Cloning Vectors
 Cosmids combine essential elements of a plasmid
and Lambda systems; a small plasmid that
contains the cos (cohesive termini) sites from
phage DNA, a plasmid origin of replication, and
genes for antibiotic resistance.
 Packaged into a bacteriophage protein coat, but
Shown above is a 50,000 base-pair
long DNA molecule bound with six
EcoRI molecules, and imaged using
the atomic force microscope. This
image clearly indicates the six EcoRI
"sites" and allows an accurate
restriction enzyme map of the cosmid
to be generated.

the plasmid replicates like a plasmid instead of http://

homer.ornl.gov/cbps/afmimaging.htm

phage DNA once it is in the bacteria.

 Fragments from 30 to 46 kb can be
accommodated by a cosmid vector.
 Recombinant cosmids are packaged into lambda
caspids
 Recombinant cosmid is injected into the bacterial
cell where the rcosmid arranges into a circle and
replicates as a plasmid. It can be maintained and
recovered just as plasmids.
Yeast Artificial Chromosomes(YACs)
 YACs can hold up to 500 kbs.
 YACs are designed to replicate as
plasmids in bacteria when no foreign
DNA is present. Once a fragment is
inserted, YACs are transferred to cells,
they then replicate as eukaryotic
chromosomes.
 YACs contain: a yeast centromere, two
yeast telomeres, a bacterial origin of
replication, and bacterial selectable
markers.
 YAC plasmidYeast chromosome
 DNA is inserted to a unique restriction
site, and cleaves the plasmid with
another restriction endonuclease that
removes a fragment of DNA and causes
the YAC to become linear. Once in the
cell, the rYAC replicates as a
chromosome, also replicating the
foreign DNA.
 Mammalian Cell Vectors
Mammalian cells are used because bacteria are not able to produce complex
eukaryotic proteins that are modified by processes such as glycosylation, or if
the mRNA needs to be processed after transcription.
There are several mammalian cell vectors:
 Simian virus 40 (SV40) - a small DNA tumor virus, could only hold a small
piece of DNA and caused only transient (temporary) expression of the
inserted DNA.
 Retrovirus- a single-stranded RNA virus that contains a gene for the enzyme
reverse transcriptase to create double-stranded DNA from RNA template, so
that the DNA can integrate into the host cell’s genome. It needs to infect
actively dividing cells.
 Adenovirus- a double-stranded DNA virus that can infect many types of host
cells with high efficiency, with a low chance for causing disease. It does not
have to infect actively dividing cells.
 DNA Cloning

There are several steps involved in cloning a gene in a cell. The

specific steps in an individual procedure may vary, but most follow
these steps:

Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
 Creating Recombinant DNA
A plasmid vector is digested with EcoRI at a single site to
produce two sticky ends.

A sample of human DNA is also digested with EcoRI to

produce pieces with the same sticky ends.

The two samples are mixed and allowed to hybridize, some

molecules will form with pieces of human DNA inserted into the p
lasmid vector at the EcoRI site.

DNA ligase is used to covalently link the fragments.

Chemical Reaction of DNA Ligase

 Phosphodiester bond (covalent bond)

 ATP, NAD+
 DNA Cloning

There are several steps involved in cloning a gene in a cell. The

specific steps in an individual procedure may vary, but most follow
these steps:

Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
 Inserting vectors into host cells

Transformation: cell made competent to take up DNA

Transduction: when the cloning vector used has aspects of a
virus, the host cell can be infected (transfected) to insert the
recombinant molecule
Electroporation: the cell is placed in an electric field such
that small pores are temporarily opened in the membrane.
Added DNA can enter through these pores.
Microinjection of DNA in the cell nucleus, such as an animal
egg, is needed to introduce DNA into an entire animal. The
DNA integrates into the animal chromosomes, the egg is
implanted, and the animal is born with the desired traits.
http://plantandsoil.unl.edu/croptechnology2005/crop_tech/animationOut.cgi?anim_name=bacteria_transformation.swf
http://www.phschool.com/science/biology_place/labbench/lab6/images/trananim.gif
Microinjection
 DNA Cloning

There are several steps involved in cloning a gene in a cell. The

specific steps in an individual procedure may vary, but most follow
these steps:

Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Some possible ligation reaction products:

Recombinant No insert Fragments No ligation

To select host cells containing recombinant DNA

Phenotyping
Colony hybridization
To selectively kill cells, and select for successfully transformed
bacterial cells, you must screen the original bacterial colonies
from the original master plate, and make exact copies by
replica plating :

• A sterile velveteen pad is placed on the plate, with cells

sticking to it.
• The pad is pressed on media in other plates, creating exact
replicas of the original plate
• Antibiotics in the media in the new plates will kill any
bacteria that do not have the recombinant plasmid inside of
them.
• The original plate is then examined to determine which
bacterial colonies were successfully transformed.
Some possible products of the transformation reaction:

Bacterial cell Genomic DNA

Plasmid with insert Plasmid w/o insert No plasmid

Ampicillin resistant Ampicillin resistant No ampicillin
resistance
Tetracycline sensitive Tetracycline
resistance No tetracycline
resistance
Finding the right clone by colony hybridization

In the following scheme, bacterial containing recombinant

plasmids are grown as clones.

The clones are blot transferred to a membrane sheet, and the DNA
present denatured and fixed onto the surface.

Adding a radioactive "probe" or complementary fragment and

allowing the DNA to hybridize followed by exposure to X-ray film id
entifies the clone containing recombinant DNA with the correct inser
t.
Colony hybridization - to identify clone containing gene of interest
Propagation
 Once colonies are
identified, they are
cultured in broth to
increase numbers and
therefore the amount
of DNA
 Samples are also
prepared for storage
at -80 degrees. They
can be kept for many
years this way.
A cloned DNA fragment can be replicated inside a bacterial cell
 Bacterium Cell containing gene
1 Gene inserted of interest
into plasmid

Bacterial Plasmid Gene of

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

Recombinate
bacterium
3
3 Host cell grown in culture,
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest Protein expressed
by gene of interest
Copies of gene Protein harvested

Basic Basic
research 4 Basic research and research
on gene various applications on protein

Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth