Gene Cloning

Cloning - a definition
 From the Greek - klon, a twig
 An aggregate of the asexually produced
progeny of an individual;a group of replicas of
all or part of a macromolecule (such as DNA
or an antibody)
 An individual grown from a single somatic cell
of its parent & genetically identical to it
 Clone: a collection of molecules or
cells, all identical to an original
molecule or cell
 DNA CLONING
A method for identifying and
purifying a particular DNA fragment
(clone) of interest from a complex
mixture of DNA fragments, and then
producing large numbers of the
fragment (clone) of interest.
 Gene cloning

 When DNA is extracted

from an organism, all its
genes are obtained
 In gene (DNA) cloning
a particular gene is
copied (cloned)
 Why Clone DNA?
 A particular gene can be isolated and its
nucleotide sequence determined
 Control sequences of DNA can be identified &
analyzed
 Protein/enzyme/RNA function can be
investigated
 Mutations can be identified, e.g. gene defects
related to specific diseases
 Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
Figure 20.2a

Gene Cloning & Expression with Plasmid Vector

Bacterium
1 Gene inserted into
Cell containing
plasmid
gene of interest

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

Recombinant
bacterium
Figure 20.2b

3 Host cell grown in

culture to form a clone
of cells containing the
“cloned” gene of interest
Gene of Protein expressed from
interest gene of interest
Copies of gene Protein harvested

4 Basic research
Basic and various Basic
research applications research
on gene 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
Sources of DNA for Cloning
 1) Chromosomal DNA

 2) Extrachromosomal DNA

 3) RNA converted to cDNA

 4) PCR-amplified DNA
Separation & Visualization of DNA
Fragments
RNA converted to cDNA
PCR-amplified DNA
The 3 General Steps Used to Clone DNA
with vector

 Isolate DNA from an organism

 Cut the organismal DNA and the
vector with restriction enzymes
making recombinant DNA
 Introduce the recombinant DNA
into a host
Cloning Tools
 Restriction endonucleases
 Ligase

 Vectors

 Host

 Methods for introducing DNA

into a host cell
 Restriction Enzymes
 Recognize a specific site (generally a
pallindromic sequence)
 Produce overhangs or straight cuts
 Naturally found in bacteria, they protect
against viruses and foreign DNA
 More than 400 enzymes have been isolated
 Named for the organism they from which
they are isolated
The first letter is that of the genus and
the 2nd and 3rd are from the species
Nomenclature
 EcoRI

 E = genus
(Escherichia)
 co = species
(coli)
 R = strain

 I = # of
enzyme
Figure 20.3-1
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3 5
5 AATTC 3
G
CTTAA G
5 Sticky 3
3 5
end
Figure 20.3-2
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3 5
5 AATTC 3
G
CTTAA G
5 Sticky 3
3 5
end
5
AATTC 3
G G
CTTAA
2 DNA fragment added 3
from another molecule 5
cut by same enzyme.
Base pairing occurs.

5 3 5 3 5 3
G AATT C G AATT C
C TTAA G C TTAA G
3 53 5 3 5
One possible combination
Figure 20.3-3
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
backbones.
3 5
5 AATTC 3
G
CTTAA G
5 Sticky 3
3 5
end
5
AATTC 3
G G
CTTAA
2 DNA fragment added 3
from another molecule 5
cut by same enzyme.
Base pairing occurs.

5 3 5 3 5 3
G AATT C G AATT C
C TTAA G C TTAA G
3 53 5 3 5
One possible combination
3 DNA ligase
seals strands
5 3

3 Recombinant DNA molecule 5

Cloning vectors

allowing the exogenous DNA to be

inserted, stored, and manipulated
mainly at DNA level.
1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
4 BACs & YACs
Plasmid vectors
Plasmid vectors are double-stranded, circular, self-
replicating, extra-chromosomal DNA molecules.
 Advantages:
 Small, easy to handle

 Straightforward selection strategies

 Useful for cloning small DNA fragments

(< 10kbp)
 Disadvantages:
 Less useful for cloning large DNA fragments

(> 10kbp)
 A plasmid vector for cloning

1. Contains an origin of replication, allowing

for replication independent of host’s
genome.
2. Contains Selective markers: Selection of
cells containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Must have:
1) Ori
2) A dominant selectable
Marker
3) Cleavage sites for cloning
4) (high copy no.)
 Bacteriophage vectors

 Advantages:
 Useful for cloning large DNA
fragments
(10 - 23 kbp)
 Inherent size selection for
large inserts
 Disadvantages:
 Less easy to handle
 vectors  Left arm:
 head & tail
proteins
 Right arm:
 DNA synthesis

 regulation

 host lysis

 Deleted central
region:
 integration &
excision
 regulation
Cosmid vectors
combine the properties of plasmid vectors with
the useful properties of the l cos site
 Advantages:
 Useful for cloning very large DNA
fragments
(32 - 47 kbp)
 Inherent size selection for large
inserts
 Handle like plasmids
 Disadvantages:
 Not easy to handle very large plasmids
(~ 50 kbp)
 BACs and YACs
BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
 Advantages:
 Useful for cloning extremely large DNA
fragments
(100 - 2,000 kbp)
 This is very important for genome
sequencing projects
 Disadvantages:
 Not easy to handle extremely large DNA
molecules
BAC vector

 oriS and oriE

mediate replication
 parA and parB
maintain single
copy number
 ChloramphenicolR
marker
 YAC vector
large
inserts

ARS URA3 HIS3

telomere centromere markers telomere

replication
origin

 Capable of carrying inserts of 200 - 2000 kbp

in yeast
 What determines the choice vector?

 insert size
 vector size
 restriction sites
 copy number
 cloning efficiency
 ability to screen for inserts
 what down-stream experiments do you plan?
Expression vector
 Btech10

expression vector pSE420

• polylinker: insert desired

DNA
• amp resistance
• trc promoter
• lacO (operator)
• Shine-Dalgarno (S/D) site
(ribosome binding)
• T1, T2 transcription
terminators
• lacI (lac repressor)
(Fig. 31.4, p. 1002, Madigan et al.)

cloned gene expressed;

growth inducer added
product produced
 Host organism
 bacterial host – E. coli
 eukaryotic host – yeast
(Saccharomyces cerevisiae)
 other hosts – other yeasts, insect
cells, etc.
Method of introducing DNA into
the host cell

Transformation into the host cell

• bacterial cells take up naked DNA molecules

• cells are made “competent”

• cells treated with ice-cold CaCl2 then heat-

shocked

• efficiency of 107 to 108 transformed

colonies/μg DNA

• maximum transformation frequency of 10-3

 Method of introducing DNA into
the host cell
Electroporation of the DNA into the host cell
• “electric field-mediated

http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg
membrane permeabilization”

• high strength electric field in

the presence of DNA

• protocols differ for various

species

• efficiencies of 109 per μg DNA

(3 kb) & 106 (136 kb)
 Method of introducing DNA into
the host cell
Conjugation
• natural transmission from donor to recipient

• host cell that is not readily transformed

• form cell to cell junctions

Transfection of the DNA
• DNA is packaged in vitro into phage particles

• phages are allowed to infect bacterial cells

• term also used in DNA transfer to eukaryotic cells

• DNA is transiently expressed

 Btech8

Other methods for introducing DNA

microprojectile “gun”
  Methods for selecting and/or screening cells that
carry the inserted foreign DNA

Selection refers to application of conditions that

favors the growth of cells or phages that carry
the vector or vector and insert.

• antibiotic resistance

• nutrient requirements

• plaque formation
Screening allows all cells to grow, but tests the
resulting clones for the presence of the insert in
the vector.

• antibiotic resistance/sensitivity

• nutrient requirements

• plaque type

• blue-white selection (β-galactosidase)

• specific (hybridization, antibodies, PCR)

 Screening of the clone
 The medium in this
petri dish contains the
antibiotic Kanamycin
 The bacteria on the
right contain Kanr, a
plasmid that is
resistant to Kanamycin,
while the one on the
left has no resistance
 Note the difference in
growth
 Blue/White Color Screening

lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product
Selecting Colonies with
Recombinant Plasmids
 Colony hybridization

Radioactive
Labeling of DNA
Screening for specific
cDNA plasmids in a cDNA
Library by using an antibody probe
The antibody binds to a specific
Site on a protein that is made via
The inserted foreign DNA. This
Is a Western Blot since it uses antibody
To detect a protein.
 DNA Libraries

 A DNA library is a collection of clones

of DNA designed so that there is a high
probability of finding any particular
piece of the source DNA in the
collection.
 Types of DNA Libraries

 The genomic library contains DNA

fragments representing the entire
genome of an organism.
 The cDNA library contains only
complementary DNA molecules
synthesized from mRNA molecules in a
cell.
Figure 8.7 Use of partial digestion with a restriction enzyme to produce DNA
fragments of appropriate size for constructing a genomic library.
Figure 20.5

Foreign genome

Cut with restriction enzymes into either

small or large Bacterial artificial
fragments fragments chromosome (BAC)
Large
insert
with
many
genes

Recombinant (b) BAC clone

plasmids

Plasmid
clone

(a) Plasmid library (c) Storing genome libraries

cDNA Libraries are best for eukaryotes

Figure 8.8 The synthesis of double-stranded complementary DNA (cDNA) from a

polyadenylated mRNA, using reverse transcriptase, RNase H, DNA polymerase I,
and DNA ligase.
Figure 20.6-1
DNA in
nucleus
mRNAs in
cytoplasm
Figure 20.6-2
DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand
Figure 20.6-3
DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand

5 A A A A A A 3
3 T T T T T 5
Figure 20.6-4
DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand

5 A A A A A A 3
3 T T T T T 5

5 3
3 5
DNA
polymerase
Figure 20.6-5
DNA in
nucleus
mRNAs in
cytoplasm

Reverse
transcriptase Poly-A tail
mRNA
5 A A A A A A 3
3 T T T T T 5
DNA Primer
strand

5 A A A A A A 3
3 T T T T T 5

5 3
3 5
DNA
polymerase

5 3
3 5
cDNA
End

Sayonara ………