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 35-45 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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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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 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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Lambda vector

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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 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

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

f. A collection of 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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CONSTRUCTION OF GENOMIC LIBRARY

GENE CLONING

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

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