Genetics Engineering:

CLONING

Dyah Ayu Oktavianie, DVM., M.Biotech

PKH-UB

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Outline
1. Key Concepts
2. DNA Cloning
3. Genetic Engineering
4. Key Terms
5. Conclusions

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Key Concepts

Genetic experiments have been

proceeding in nature for billions of years

Genetic changes are brought about by

Recombinant DNA technology

With technology, researchers can isolate,

cut, and splice together gene regions
from different species, and amplify the
number of copies
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Key Concepts

Recombinant DNA technology depends on 3

activities

Cutting DNA into fragments

Insertion of fragments into cloning tools like plasmids

clone and identification of desired clone

Genetic engineering involves isolating,

modifying, and inserting genes back into the
same organism or into a different one

Social, ethical, legal, and ecological questions

are raised by the new technology
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Glowing mice

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Molecular Cloning
MCS
Bacterial
plasmid vector
Origin of replication

Multiplicity

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Gene Cloning
Isolation and amplification of an individual
gene sequence by insertion of that
sequence into a cells where it can be
replicated
Involves the construction of novel DNA
molecules by joining DNA from different
sources
Product is Recombinant DNA (rDNA)
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Basic Events in Gene Cloning

Isolation and amplification of gene of interest
Incorporate gene into a vector (small replicating
DNA molecule, usually circular)
Introduce recombinant vector into host cell via
transformation
Select for the cells that have acquired the
recombinant DNA molecule
Multiply recombinant vector within host cell to
produce a number of identical copies of the
cloned gene
Extract the vector to obtain the copy of the gene
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Components of Gene Cloning

Vectors (cloning vehicles)
Enzymes for cutting and joining the
DNA fragments
The DNA fragments (Target DNA)
Selection process

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Cloning Vectors
Requirements of a vector to serve as a carrier
molecule

The choice of a vector depends on the design of

the experimental system and how the cloned gene
will be screened or utilized subsequently
- host targets
- size of DNA fragments
- screening methods

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 Most vectors contain a prokaryotic origin of

replication allowing maintenance in bacterial cells
Some vectors contain an additional eukaryotic
origin of replication allowing autonomous, episomal
replication in eukaryotic cells.
Multiple unique cloning sites are often included for
versatility and easier library construction.

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 Antibiotic resistance genes and/or other

selectable markers enable identification of
cells that have acquired the vector construct.
Some vectors contain inducible or tissuespecific promoters permitting controlled
expression of introduced genes in transfected
cells or transgenic animals.
Modern vectors contain multi-functional
elements designed to permit a combination of
cloning, DNA sequencing, in vitro
mutagenesis and transcription and episomal
replication.

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Vectors
Must contain a replicon that enables it to
replicate in host cells (region of DNA that is
amplified, i.e.: has origin of replication)
Small enough and unlikely to degrade during
purification.
Several marker genes
Unique cleavage site(s)
For expression, must contain control elements,
such as promoters, terminators, ribosome
binding sites, etc
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Types of Vectors

Plasmids
Cosmids
Fosmids
Phages
Yeast Artificial Chromosomes (YACs)
Transposons
Bacterial Artificial Chromosomes (BACs)
Viruses

retroviruses
adenoviruses
adeno-associated viruses
herpes simplex virus
rhinoviruses
Human Immunodeficiency Virus (HIV)
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APPROXIMATE MAXIMUM LENGTH DNA

THAT CAN BE CLONED IN VECTORS
Vector Type
Plasmid
phage
Cosmid
P 1 phage
BAC (bacterial artificial chromosom)
YAC (yeast artificial chromosom)

Cloned DNA ( kb )
10
25
45
100
300
1000

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Choice of vector
Depends on nature of protocol or
experiment
Type of host cell to accommodate rDNA
Prokaryotic
Eukaryotic

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Plasmids vector
Double stranded, circular DNA which exist in
bacteria.
May exist as single copy per cell or multi-copy per
cell (10-20 genomes/cell), or even under relaxed
replication control where up to 1000 copies/cell can
be maintained
Size of rDNA insertions limited to ~10kb
Covalently closed, circular, double stranded DNA
molecules that occur naturally and replicate
extrachromosomally in bacteria
Many confer drug resistance to bacterial strains
Origin of replication present (ORI)
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 Interruptable gene encoding for enzyme beta

galactosidase (lacZ)
Polylinker resides in the middle
Enzyme activity can be used as marker
for gene insertion
Disrupted gene = nonfunctional
Intact gene = functional
Media containing XGAL chromagenic
substrate used (blue colonies = intact;
white colonies = disrupted)
Amp resistance gene still present (= beta
lactamase), Tet resistance gene omitted
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General Cloning Scheme

Vector and foreign gene to be inserted are
purified/modified separately before ligating the two
together
Ligated products are introduced into competent
bacterial cells by transformation techniques.
Individual colonies are analyzed separately.
Vectors able to survive under antibiotic selection
are amplified in bacterial hosts by autonomous
replication
Plasmid DNA containing the gene of interest is
purified from large scale cultures

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Cosmid vectors
Hybrid molecules containing components of both
lambda and plasmid DNA
Lambda components: COS sequences (required for
in vitro packaging into phage coats)
Plasmid DNA components: ORI + Antibiotic
resistance gene
Cloning sites will be part of vector
rDNA is packaged using extracts of coat and tail
proteins derived from normal lambda components
BUT cannot be packaged after introduced into host cell
because rDNA does not encode the genes required for
coat proteins
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 After infection of E. coli, rDNA

molecules replicate as plasmids
Very large inserts can be
accommodated by cosmids (up to 3545 kbp)

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Cosmids
Plasmid vectors that contain a
bacteriophage lamda cos site
The cos site results in efficient packaging
of lamda DNA into virus particles
With the cos site, larger DNA inserts are
possible (up to ~40 kb)

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Page 25

Bacteriophage Vectors
Viruses that attack specific bacteria
Must first deactivate lysogenic growth
component of phage (phage DNA inserts into
host DNA, creating prophage)
Allow lytic growth cell death after infection and
replication. Cell death revealed as plaques
Insert rDNA into phage (usu. up to 25kb)
Infect bacteria with phage
Infected bacteria form plaques
Advantage: Transformation, selection very easy
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Bacteriophages

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Lambda vector

Bacteriophage lambda infects E. coli

Double-stranded, linear DNA vector suitable for library
construction
Can accommodate large segments of foreign DNA
Central 1/3 = stuffer fragment
Can be substituted with any DNA fragment of similar
size without affecting ability of lambda to package itself
and infect E. coli
Accommodates ~15kbp of foreign DNA
Foreign DNA is ligated to Left and Right Arms of
lambda Then either:
1) Transfected into E. coli as naked DNA, or
2) Packaged in vitro by combining with phage
protein components (heads and tails) (more efficient,
but labor intensive and expensive)
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Page 29

Yeast Artificial Chromosome (YAC)

Artificially produced mini chromosome,
consist of:
Centromere: important in cell division
Telomeres: Mark the end of chhromosome.
Origin of replication,
Marker genes

Able to accommodate very large inserts

(~1,000 2,000 kb)
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Bacterial Artificial Chromosome

(BAC)
Based on the naturally-occuring F plasmid
in E. coli.
F plasmid is relatively large.
Have larger capacity to accepting inserted
DNA.
Able to clone up to 300kb DNA fragments

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Producing Restriction
Fragments
Restriction enzymes
Cut at specific nucleotide sequences
Some create Sticky Ends
DNA fragments cut with the same restriction

enzyme will base-pair to form recombinant

fragments

DNA ligase
Seals nicks where fragments base pair
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Using a restriction
enzyme and DNA ligase
to make recombinant
DNA

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DNA Cloning and Genome (DNA) library

a. Restriction enzyme
cuts chromosomal or
cDNA

c. DNA or
cDNA
fragments

e. DNA fragments
and
modification
enzymes
are mixed
together

f. A collection of
recombinant
plasmids

d. Plasmid
b. Same enzyme
cuts plasmid DNA fragments
g. Host cells able
to divide rapidly
take up
recombinant
plasmids

The original plasmid is called a cloning

vector
(taxi for delivering foreign DNA into
a bacterium)

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Human gene Cloning

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widya-ugm

Constructing Genomic and cDNA

Libraries

Definition
A cloned set of rDNA fragments representing
either the entire genome of an organism
(Genomic library) or the genes transcribed in a
particular eukaryotic cell type (cDNA library)
rDNA fragments generated using restriction
endonucleases
rDNA fragments ligated to appropriate cloning
vector

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Genomic libraries
Commonly bacteriophage lambda used as
the vector
Stuffer fragment removed and replaced with 1517kbp fragments of library

Cosmids and YACs may also be used as

vectors
Contains at least one copy of all DNA
fragments in the complete library
Screened using nucleic acid probes to identify
specific genes
Subcloning is usually necessary for detailed
analysis of genes
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GENE CLONING

CONSTRUCTION OF GENOMIC LIBRARY

1. Source of DNA
2. Enzyme : restriction endonucleases
3. Vector
4. Host

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SOURCE OF DNA
Isolated from the target organism (in
which the genomics library will be
directed)
Decided the appropriate isolation method
Prepared in high purity
Fragmented using restriction
endonuclease enzymes

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Common steps involved in isolating a

particular DNA fragment from a complex
mixture of DNA fragments or molecules
1.

2.

DNA molecules are digested with enzymes called

restriction endonucleases which reduces the size
of the fragments Renders them more
manageable for cloning purposes
These products of digestion are inserted into a
DNA molecule called a vector Enables desired
fragment to be replicated in cell culture to very
high levels in a given cell

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QUIZ
Jika anda sebagai seorang peneliti akan
melakukan cloning gen penyandi protein
membran bakteri Salmonella sp. dengan
ukuran sebesar 8kb, dan bakteri
rekombinan akan ditumbuhkan dalam
medium seleksi yang mengandung
ampicillin jelaskan bahan dan metode yang
akan anda lakukan.....

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