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Genetics Engineering:

CLONING

Dyah Ayu Oktavianie, DVM., M.BiotechPKH-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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Page 6Multiplicity

Molecular Cloning

Origin of replication

MCS

Bacterial

plasmid vector

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Gene Cloning

Isolation and amplification of an individualgene sequence by insertion of thatsequence into a cells where it can be

replicated

Involves the construction of novel DNAmolecules 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 toproduce 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 theDNA fragments

The DNA fragments (Target DNA)

Selection process

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Cloning VectorsRequirements of a vector to serve as a carrier

molecule

The choice of a vector depends on the design ofthe experimental system and how the cloned genewill be screened or utilized subsequently

- host targets

- size of DNA fragments

- screening methods

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Most vectors contain a prokaryotic origin ofreplication allowing maintenance in bacterial cells

Some vectors contain an additional eukaryoticorigin of replication allowing autonomous, episomalreplication in eukaryotic cells.

Multiple unique cloning sites are often included forversatility and easier library construction.

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Antibiotic resistance genes and/or other

selectable markers enable identification ofcells that have acquired the vector construct.

Some vectors contain inducible or tissue-specific promoters permitting controlledexpression of introduced genes in transfectedcells or transgenic animals.

Modern vectors contain multi-functionalelements designed to permit a combination ofcloning, DNA sequencing, in vitro

mutagenesis and transcription and episomalreplication.

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Vectors

Must contain a replicon that enables it toreplicate in host cells (region of DNA that isamplified, i.e.: has origin of replication)

Small enough and unlikely to degrade duringpurification.

Several marker genes

Unique cleavage site(s)

For expression, must contain control elements,such as promoters, terminators, ribosomebinding 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)

APPROXIMATE MAXIMUM LENGTH DNA

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APPROXIMATE MAXIMUM LENGTH DNA

THAT CAN BE CLONED IN VECTORS

10

25

45

100

300

1000

Plasmid

phage

Cosmid

P 1 phage

BAC (bacterial artificial chromosom)YAC (yeast artificial chromosom)

Cloned DNA ( kb )Vector Type

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Choice of vector

Depends on nature of protocol orexperiment

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 percell (10-20 genomes/cell), or even under relaxedreplication control where up to 1000 copies/cell can

be maintained Size of rDNA insertions limited to ~10kb

Covalently closed, circular, double stranded DNAmolecules 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 markerfor gene insertion

Disrupted gene = nonfunctional

Intact gene = functional

Media containing XGAL chromagenicsubstrate 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 twotogether

Ligated products are introduced into competentbacterial cells by transformation techniques.

Individual colonies are analyzed separately. Vectors able to survive under antibiotic selection

are amplified in bacterial hosts by autonomousreplication

Plasmid DNA containing the gene of interest ispurified 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 phagecoats)

Plasmid DNA components: ORI + Antibioticresistance gene

Cloning sites will be part of vector

rDNA is packaged using extracts of coat and tailproteins derived from normal lambda components

BUT cannot be packaged after introduced intohost cell because rDNA does not encode thegenes required for coat proteins

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After infection of E. coli, rDNAmolecules replicate as plasmids

Very large inserts can beaccommodated by cosmids (up to 35-45kbp)

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Cosmids

Plasmid vectors that contain abacteriophage 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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Bacteriophage Vectors

Viruses that attack specific bacteria

Must first deactivate lysogenic growthcomponent of phage (phage DNA inserts intohost DNA, creating prophage)

Allow lytic growth cell death after infection andreplication. 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 libraryconstruction

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 itselfand 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 vitroby combining with phageprotein components (heads and tails) (more efficient,but labor intensive and expensive)

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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 plasmidin E. coli.

F plasmid is relatively large.

Have larger capacity to accepting insertedDNA.

Able to clone up to 300kb DNA fragments

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Producing RestrictionFragments

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 restrictionenzyme and DNA ligase

to make recombinantDNA

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a. Restriction enzymecuts chromosomal orcDNA

c. DNA orcDNAfragments

d. Plasmidfragments

e. DNA fragmentsandmodificationenzymesare mixedtogether

f. A collection ofrecombinantplasmids

g. Host cells ableto divide rapidlytake up

recombinantplasmids

b. Same enzymecuts plasmid DNA

DNA Cloning and Genome (DNA) library

The original plasmid is called a cloningvector (taxi for delivering foreign DNA into

a bacterium)

Human gene Cloning

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Human gene Cloning

widya-ugm

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widya ugm

Constructing Genomic and cDNA

Libraries

D fi i i

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Definition

A cloned set of rDNA fragments representing

either the entire genome of an organism(Genomic library) or the genes transcribed in aparticular eukaryotic cell type (cDNA library)

rDNA fragments generated using restrictionendonucleases

rDNA fragments ligated to appropriate cloningvector

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Genomic libraries

Commonly bacteriophage lambda used asthe vector Stuffer fragment removed and replaced with 15-

17kbp 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 (inwhich the genomics library will bedirected)

Decided the appropriate isolation method Prepared in high purity

Fragmented using restriction

endonuclease enzymes

Common steps involved in isolating a

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Common steps involved in isolating aparticular DNA fragment from a

complex mixture of DNA fragments or

molecules1. DNA molecules are digested with enzymes called

restriction endonucleases which reduces the sizeof the fragments Renders them more

manageable for cloning purposes2. These products of digestion are inserted into a

DNA molecule called a vector Enables desiredfragment to be replicated in cell culture to veryhigh levels in a given cell

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QUIZ

Jika anda sebagai seorang peneliti akanmelakukan cloning gen penyandi proteinmembran bakteri Salmonella sp. dengan

ukuran sebesar 8kb, dan bakterirekombinan akan ditumbuhkan dalammedium seleksi yang mengandungampicillinjelaskan bahan dan metode yangakan anda lakukan.....

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Salmonella type

Vektor ; Bakteri Enzim pemotiong dan penghubung Target DNA Penanda untuk seleksi