Gene Cloning

SHREE SIDDAGANGA COLLEGE OF PHARMACY TUMKUR, KARNATAKA SSCP TUMKUR

Content s

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History Definition Steps in cloning

o

Generating DNA fragments

o

Insertion of target DNA in to vector

o

Introduction of recombinant DNA in to host cell

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 Applications of cloning  References

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History:       

1869 - Miescher – isolated DNA from WBC. 1944 - Avery - DNA is primary genetic material. 1953 - Watson Crick - DNA double helix structure. 1962 - Arber - evidence of DNA restriction nuclease. 1966 - Nirenberg Ochoa and Khorana elucidation of genetic code. 1967 - Gellert - DNA ligase enzyme. 1972 - 73 - DNA Cloning techniques. SSCP TUMKUR

Definition: – Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism. (Sometimes called subcloning, because only part of the organism’s DNA is being cloned.) – Using nuclear DNA from one organism to create a second organism with the same nuclear DNA

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Main steps in cloning:

•Generating DNA fragments •Insertion of target DNA in to vector •Introduction of recombinant DNA in to host cell

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Generating DNA fragments :  • • • • •

Mechanical shearing: High speed mixing in blender or sonicator. Random fragment of source DNA. Does not produce 5’phasphate and 3’OH ends. Requires repair the ends (S1nuclease or DNA polymerase). Not reproducible in nature.

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 Restriction

Endonuclease Digestion: Restriction endonuclease are the enzymes isolated from bacteria that cut DNA at specific recognition nucleotide sequences known as restriction sites. • More than 200 restriction enzymes. • Size of DNA fragments depends upon restriction sites present in DNA. • It produces either sticky end or blunt ends. • Generally same enzyme is used for the vector and the DNA of interest. • Reproducibility of fragment is possible.

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- Restriction enzymes can generate both sticky ends and blunt ends after they cut the recognition sequence

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 •

Reverse Transcriptase Method: Gene organisation of eukaryotes and prokaryotes is different.

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Bacteria or yeast do not have necessary splicing mechanism for removal of introns.

•

Mature mRNA molecules from animal cell can be directly transcribed in to DNA using an enzyme reverse transcriptase.

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cDNA produced for particular protein can be joined to vector and cloned in to host cell.

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 Hybridization

Method:

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Principle: mRNA forms a complex with complementary DNA segments from which it has been transcribed.

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Total DNA from donor organism is isolated & dsDNA is converted to ssDNA by denaturation.

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Mixed with mRAN trancribed by the gene this forms DNA-RNA complex (Hybrid). SSCP TUMKUR

Cont… •

DNA-RAN complex is isolated & DNA is separated from RNA.

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ssDNA obtained can be converted to dsDNA by DNA polymerase I.

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Suitable for isolating genes which exist in multiple copies.

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 •

Chemical Synthesis: The technique is termed as reverse genetics.

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Possible only when amino acid sequence of protein of interest is known.

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For which sequence of nucleotides in mRNA & subsequently in DNA can be determined.

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This method of generating DNA fragments for cloning will be batter for creating novel proteins.

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IDENTIFICATION OF CLONE

S O U T H E R N B L O T

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Insertion of Target DNA into Vector:  Vectors:

are carrier DNAs into which foreign DNAs are spliced to make a recombinant DNA. Types of vectors: • Plasmids • Bacteriophages • Cosmids • BACs & YACs SSCP TUMKUR

Selection of Vector: VECTOR • • • • •

INSERT SIZE (kbp)

Plasmid ~ 15.0 kbp λ Phage ~ 50.0 kbp Cosmid > 50.0 <100.0 kbp Bacterial artificial ~ 300.0 kbp Chromosome (BAC) Yeast artificial ~ 2000 kbp Chromosome (YAC)

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Plasmids Plasmids are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules.

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Cont…

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Plasmids are circular pieces of DNA found naturally in bacteria.

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Plasmids can carry antibiotic resistance genes, genes for receptors, toxins or other proteins.

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Plasmids replicate separately from the genome of the organism.

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Plasmids can be engineered to be useful cloning vectors. SSCP TUMKUR

Isolation of bacterial plasmids o

Treat the prokaryotic cells with detergent.

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Treat the lysate with potassium acetate/ acetic acid solution and centrifuge.

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Treat the lysate with RNAase consequently.

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Mix the lysate with phenol. Phenol & water is separated in two layers . The water based layer contains plasmid DNA.

o

Precipitate water based layer with alcohol and collect the plasmid DNA.

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Cont…..

Ex. Of plasmids •

pBR 322- derived from E.coli prepared by Bolivar and Rodriguez.

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pAT 153 and pXf 3 are two derivatives of pBR 322 having smaller size & higher copy.

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pUC derived from E.coli initial prepared in University of California. SSCP TUMKUR

Cont…..

 Advantages

of plasmids:

– Small, easy to handle – Useful for cloning small DNA fragments (< 15kbp)

 Disadvantages

of plasmids:

– Less useful for cloning large DNA fragments (> 15kbp) SSCP TUMKUR

Bacteriophage s

• Phases has linear DNA, single break create two fragments. • Foreign DNA can be inserted between them & two fragments can be joined. • Bacteriophages act as hypodermic syringe like and insert DNA in to the host and replicates. • Lytic Cycle: rapid infection resulting in lysis of cell and release of multiple bacteriophages. •Lysogenic phages: For bacteriophage λ, DNA integrates SSCP TUMKUR in

B A C T E R I O P H A G E S SSCP TUMKUR

Cosmids • Cosmids posses properties of both plasmids and bacteriophages.

• Contain a cos site of λ phage (essential for packaging of nucleic acid into protein coat) •

plus essential features of plasmids ( plasmid origin of replication, a gene for resistance)

• Cosmids can be constructed by adding a fragment of phage λ DNA including cos site to plasmid. • A foregin DNA can be inserted into cosmid DNA

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C O S M I D S

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BACs: Bacterial Artificial Chromosomes 

The construction BAC F plasmid, which is large than other plasmid used as cloning vector.

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BACs can accept DNA inserts of around 300kb SSCP TUMKUR

YACs:Yeast Artificial Chromosomes • YAC are the most sophisticated yeast vectors. • They have centromeric & telomeric region of chromosome. • very large piece of DNA can be cloned (particular human DNA) ~ 2000 kbp. SSCP TUMKUR

Ligation • Ligation is the process of joining two pieces of DNA from different sources together through the formation of a covalent bond.

• DNA ligase is the enzyme used to catalyze this reaction.

• These enzymes are originally isolated from

viruses, also occur in E.coli & eukaryotic cell.

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DNA Ligases Type E. coli ligase T7 ligase

T4 ligase

Energy source NAD (turned into AMP and NMN) ATP (turned into AMP and PPi) ATP (turned into AMP and PPi) SSCP TUMKUR

Used for: Sticky ends

Sticky ends

Both sticky and blunt ends

PRECAUTION • The most important precaution prior to setting up a ligation reaction is to ensure that the cut vector is prevented from recircularization. • This is achieved by digesting the plasmid by the same restriction enzyme(s). • Treatment with alkaline phosphatase which removes the terminal phosphate groups from the cut ends of the plasmid. This ensures that the cut plasmid does not recircularize. SSCP TUMKUR

C O H E S I V E L I G A T I O N SSCP TUMKUR

BLUNT END LIGATION The blunt end DNA can be joined by :  Homopolymer

tailing

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Use of Linkers Molecule



Use of Adaptors Molecule

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Homopolymer tailing

• The complementary DNA strands can be joined together by annealing.

• Addition of oligo (dA) to 3’ ends of some

DNA molecule and addition of oligo (dT) to 3’ ends of other molecule.

• The homopolymer extension can be synthesized by using terminal deoxynucle- otidyltrasferase (calf thymus). SSCP TUMKUR

Homopolymer tailing Insert

Vector

3’

5’ 3’

dATP

5’

3’AA

3’ 5’

5’ 3’

5’

Terminal deoxynucleotidyltransferas e

AAA

5’

3’

5’

3’

A LIGATION SSCP TUMKUR

TTT

dTTP

TTT 5’

3’

Use of Linkers Molecule • Linkers are chemically synthesized, short, double strand DNA molecules.

• possess restriction enzyme cleavage sites. • they can be ligated to blunt ends of any DNA. • Cut with specific restriction enzymes to produce sticky end DNA fragments SSCP TUMKUR

Blunt- ended insert 5’ 3’

3’ 5’

Linker (e.g. Bam HI) 5’ P GGGATCCC OH 3’ 3’ OH CCCTAGGG P 5’

Ligate to linker GGGATCCCGGGATCCC CCCTAGGGCCCTAG GG

GATCCC GG

CCCTAGGGCCCTAGG G GGGATCCCGGGATC CC

Cut with Bam HI GG CCCTAG

Ligate to vector cut with Bam HI SSCP TUMKUR

Use of Adaptors Molecule • Adaptors are chemically synthesized, short, double-stranded DNA molecules.

• Adaptor contains preformed sticky or cohesive ends.

• They are useful to be ligated to DNA fragment with blunt ends.

• The DNA fragments held to adaptors are finally ligated to vector DNA molecules. SSCP TUMKUR

5’ 3’

GGGATCCCGGGATCCC CCCTAGGG 3’

5 DNA Fragment ’

GGGATCCCGGGATC CC CCCTAGG G

+

CCCTAGGG GGGATCCCGGGATCCC Adaptors

DNA ligase+ATP

CCCTAGGG GGGATCCCGGGATCCC

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Introduction of recombinant DNA in to host

cell • After creating new vector construct it needs to

insert into a host cell so that it can be replicate.

• Hosts are the living systems or cells in which vector can be propagated

• The process of introducing the foreign DNA into the host cell is called transformation.

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Cont…

• Host cell may be prokaryotic (bacteria) or eukaryotic (fungi, animal, plant).

• Micro organisms are preferred as host cells, since they multiply faster.

PROKARYOTIC HOST (Bacteria): • Not every bacterial cell is able to take up plasmid DNA.

• Bacterial cells that can take up DNA from

the environment are said to be competent.

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Escherichia coli:

• E.coli was the first organism used in the DNA technology experiments.

• Is common Gram – ve bacterium of human and animal intestine.

• Under suitable environment E.coli

multiply two times in every 20 min. Limitations of E.coli: - Formation of endotoxins that are toxic. -

Cannot perform post- translational modification SSCP TUMKUR

Eukaryotic hosts:

• Eukaryotic organisms are preferred to produce human proteins.

• Most commonly used organism is yeast, Saccharomyces.

• It is non pathogenic organism rutinely used in baking industry. Mammalian cells: - Certain complex protein produced by mammalian cells Eg. Tissue plasminogen activator. -

mammalian cell possess post translation SSCP TUMKUR modifications.

Some Examples of Host cells Group

Examples

Prokaryotic Bacteria

E.coli Bacillus Subtilis Streptomyces sp

Eukaryotic Fungi Animals

Plants

Saccharomyces cervisiae Aspergillus nidulans Insect cells mammalian cells Whole organisms Protoplasts Intact cell SSCP TUMKUR

Method of gene transfer

Introduction of foreign DNA in to host cell is important in gene cloning, commonaly employed methods are:

o Transformation o Lipofection o Transduction o Electroporation

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Transformionat • Is a method of introducing foreign DNA in to bacterial cells (E.coli)

• Carried out in ice-cold CaCl2 (0-5c) & subsequent heat shock (37- 45c for about 90 sec.)

• Some times calcium phosphate or

diethyl aminoethyl dextran (DEAEdextran) is replaced to CaCl2 . SSCP TUMKUR

Lipofection The liposome mediated gene transfer, is referred as lipofection. • It is vary efficient technique & is used for -

bacteria, animal & plant host cell.

• On treatment of DNA fragment with liposome,

the DNA get encapsulated inside liposome.

• Liposome can adhere to cell membrane &

transfer DNA fragments.

• DNA enters the cell & then to the nucleus.

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Transduction  The

foreign DNA can be packed inside animal viruses.

 These

viruses can naturally infect the cells & introduce the DNA into host cell.

 The

transfer of DNA by this approach is referred as transduction.

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Electroporation  Electroporation

is technique involves electric field mediated membrane permeabilization.

 It

is based on the principle that high voltage pulses can induce cell plasma membrane fuse.

 Electric

shock induce cellular uptake of exogenous DNA from the suspending solution.

 It

is simple & rapid technique for introducing genes in to the cells of various organisms.

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WHY CLONE A GENE? 

A particular gene can be isolated and its nucleotide sequence can be determined

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Coding regions and control elements like promoters can be identified and analyzed

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Protein/enzyme/RNA function can be investigated

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Mutations can be identified, e.g. gene defects related to specific diseases

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Cont….  Organisms

can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.

 If a protein is not abundant naturally, its gene can be cloned to produce the protein in large amounts

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References :  Gene

biotechnology by S. N. Jogdand.

 Biotechnology  Molecular

Alberts.

 www.slide

by U. Satyanarayana.

Biology of Cell by Bruce world.com.

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