RECOMBINANT DNA

T H YTECHNOLOGY

What is Genetic What is Genetic Engineering?Engineering?Engineering?Engineering?

• Genetic engineering (GE) is the transfer of g g ( )genes from one organism to another through means that do not occur in nature, but through human intervention. atu e, but t oug u a te e t oThis involves isolating and then moving genes within and without different species by recombinant DNA techniques and other by recombinant DNA techniques and other manipulation of the genetic construct outside the traditional practices such as sexual and asexual breeding sexual and asexual breeding, hybridization, fermentation, in-vitro fertilization and tissue culture.

The Term of Gene The Term of Gene ManipulationManipulation

l d f d hGene Manipulation is defined as the formation of new combinations of heritable material by the insertion of heritable material by the insertion of nucleic acid molecules，produced by whatever means outside the cell, into any whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation Also capable of continued propagation. Also named gene cloning.

DNA Manipulation•Gene cloning is production of many identical copies of the 

DNA Manipulation

same gene.•If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product.g p p•Cloned genes have many research purposes: determining the base sequence between normal and mutated genes altering the phenotype obtaining themutated genes, altering the phenotype, obtaining the protein coded by a specific gene, etc. •Humans can be treated with gene therapy: alteration of the phenotype in a beneficial way 

DNA ManipulationDNA Manipulation• Recombinant DNA (rDNA) contains DNA from two or more

different sources

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– Requires:• A vector

– introduces rDNA into host cell– Plasmids (small accessory rings of DNA from bacteria) are

common vectors– Phage vectors (bacterial viruses) can also be used

• Two enzymes to introduce foreign DNA into vector DNA– A restriction enzyme - cleaves DNA

– Bacterial enzyme that stops viral reproduction by cleaving viral DNA

– Act as molecular scisssors (cut plasmids and foreign ( p ghuman DNA)

– Produce short single stranded “sticky ends” where insertions of foreign DNA can be made

– A DNA ligase enzyme - seals DNA into an opening created

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g y p gby the restriction enzyme

Cloning a Cloning a Cloning a Cloning a Human GeneHuman Gene

Restriction enzyme EcoRIRestriction enzyme EcoRI–Bacterial enzyme that stops viral reproduction by cleaving viral DNA–Act as molecular scisssors (cut plasmids and foreign human DNA)P d h t i l t d d “ ti k d ”

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–Produce short single stranded “sticky ends” where insertions of foreign DNA can be made

What is a Gene? a What is a Gene? a Genome?Genome?Genome?Genome?

• A gene is a unit of inheritance. For example children tend to look like their example, children tend to look like their parents. We inherit our features through our genes, half of which come from one parent and half from the other parent, and half from the other.

• A gene is also defined by a digital code of j t f DNA b (A T G & C) th t i just four DNA bases (A,T,G & C) that is nearly universal for all known life forms, whether viral, bacterial, fungal, plant, whether viral, bacterial, fungal, plant, animal or human. The average size gene of bacteria is about 1,000 bases long.

• Since genes are encoded are encoded by the DNA bases that

i th comprise the linear strands of a chromos-of a chromosome, the genes are

d i arranged in linear order along chrom-gosomes, and they can be mapped mapped.

Wh Clone DNA?Wh Clone DNA?Why Clone DNA?Why Clone DNA?• A particular gene can be isolated and its

l tid d t i dnucleotide sequence determined• Control sequences of DNA can be identified &

analyzedanalyzed• Protein/enzyme/RNA function can be

investigatedg• 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 etcresistance, etc.

How is DNA cloned?How is DNA cloned?

Cell-based DNA cloning Cell-free DNA cloning (PCR)

Clone in dividing cellsClone in dividing cellsClone in dividing cellsClone in dividing cells

Clone Clone ininininPPCCCCRR

Restriction Endonucleases--The Molecular Scissors--The Molecular Scissors

Host enzymes that prevent the invasion of foreign DNAs such as viral DNA by cutting them upDNAs such as viral DNA, by cutting them up.

These enzymes cut within the foreign DNAs, rather

Restriction

y g ,than chewing them away from the ends.

EndonucleasesThese enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands GAATTCSome enzymes make staggered cutsGAATTC

CTTAAG

Some make even cuts CCCGGGGGGCCC

Sticky end

Sticky end

DNA ligase covalently links two DNA strands

Restriction

5’ 3’

Restrictionenzyme

Ligaseg

5’3’

Restriction

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Restrictionenzyme

Ligase

Plasmid: a cloning vector or Plasmid: a cloning vector or h lh lvehiclevehicle

•Restriction Enzyme Mechanisms:• Preparation of DNAs to be joined• (a)Staggered cut: leaves “sticky • (a)Staggered cut: leaves sticky ends”

•Restriction Enzyme Mechanisms: P ti f DNA t b j i dPreparation of DNAs to be joined:

• (b) Blunt End

Ligation of DNA cut with a Ligation of DNA cut with a R t i ti ER t i ti ERestriction EnzymeRestriction Enzyme

•Staggered “sticky ends”•Staggered sticky ends

Ligation of DNA cut with a Ligation of DNA cut with a R t i ti ER t i ti ERestriction EnzymeRestriction Enzyme

Role of T4 DNA Ligase•Role of T4 DNA Ligase

Selectable Markers:Selectable Markers:Selectable Markers:Selectable Markers:

cDNA LibrarycDNA Libraryyy• Used to obtain

functional eukaryotic coding regions.E li d t • E. coli does not process introns.

• First step: • First step: Isolate poly A+ mRNA with oligo (dT) cellulose

cDNA Library preparation

Cloning vectorsCloning vectors

• Cloning vectors are carrier DNA molecules. Four important f f ll l h hfeatures of all cloning vectors are that they:

• (i) can independently replicate themselves and the foreign DNA segments they carry;

• (ii) contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector;

• (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host cells that do not contain a vector; and (i ) l i l f h h ll• (iv) are relatively easy to recover from the host cell.

PlasmidsPlasmids• Naturally occurring

t h l DNA extrachromosomal DNA • Plasmids are circular dsDNA • Plasmids can be cleaved by

restriction enzymes, leaving sticky y , g yends

• Artificial plasmids can be • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of fragments to the sticky ends of plasmid

Vectors -- the DNA carriersMust have a origin of replicationMust have a origin of replicationAllow the vector as well as the foreign DNA to amplify in the host cell

1) Plasmids)

2) Phage

Origin of replication

Antibiotic-resistant genesgs Allow the host to grow on

selective mediaCan selectively amplify this specific vector in thethis specific vector in the host cell

Multiple cloning sites

Allow insertion of foreign DNAllow insertion of foreign DN

PlasmidsPlasmids

• Recombinant DNA vectors:– Amplification of DNA fragment can be achieved in the Amplification of DNA fragment can be achieved in the

cell using cloning vectors: plasmid or bacteriophages– Plasmid Small circular DNA in bacteria or yeast cells

Accumulate 1-5 kb inserts

LacZ encodes β galactosidaseLacZ encodes β-galactosidaseLacl – encodes factor controling transcription of lacZ

B t i h B t i h λλ (L bd )(L bd )Bacteriophage Bacteriophage λλ (Lambda)(Lambda)•For cloning inserts of 10-20 Kbg•Plasmid libraries hold up to 10 kb insertsinserts

Bacteriophage Bacteriophage λλ (Lambda)Life (Lambda)Life C lC lCycleCycle

Lytic •Lytic Cycle:Production of progenyn of progeny

•Lysogenic lCycle:

Integration into b lbacterial chromosome

BACs: Bacterial Artificial BACs: Bacterial Artificial ChChChromosomesChromosomes

•Based on P1 bacteriophage, the F p g ,plasmid and the lacZ region of pUC plasmidsp

•It’s a low copy number plasmid•Carries 50 300kb fragments•Carries 50-300kb fragments

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomesChromosomesChromosomes

YACs:Yeast Artificial YACs:Yeast Artificial ChChChromosomesChromosomes

DNA can be inserted into a cell DNA can be inserted into a cell by:by:by:by:

• TransformationEl t ti• Electroporation

• Protoplast fusion

DNA can be inserted into a cell DNA can be inserted into a cell by:by:

• Microinjection• Gene gun

by:by:

• Gene gun

Bacterial transformationIntroduction of DNA into bacteriaSpontaneous uptake – low probabilityE. coli – cells treated with CaCl2Less than 1 of 103 cells acquire aLess than 1 of 10 cells acquire a plasmidSelection of transformed cells:

resistance to antibioticsusing chromogenic substances

Antibiotics: molecules produced by microorganism that kill othermicroorganism that kill other microorganismpeniciline, tetracycline, ciplroflaxine –inhibits gyrase in the complex with DNA

i hibit DNA li ti– inhibits DNA replication

Chromogenic substances:

ScreeningScreeningScreeningScreening• The medium in this petri p

dish contains the antibiotic Kanamycin

• The bacteria on the rightThe bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin while the oneKanamycin, while the one on the left has no resistance

• Note the difference in• Note the difference in growth

PropagationPropagationp gp g• Once colonies are

identified they areidentified, they are cultured in broth to increase numbers and therefore the amount of DNA

• Samples are also prepared for storage

t 80 d That -80 degrees. They can be kept for many years this wayyears this way.

Agricultural ApplicationsAgricultural Applicationsg ppg ppHerbicide

resistanceresistance-Broadleaf plants have been have been engineered to be resistant to the resistant to the herbicide glyphosateglyphosate

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Agricultural ApplicationsAgricultural ApplicationsPest resistance

-Insecticidal proteins have been -Insecticidal proteins have been transferred into crop plants to make them pest-resistantthem pest resistant

-Bt toxin from Bacillus thuringiensis

Golden riceRice that has been genetically -Rice that has been genetically

modified to produce β-carotene (provitamin A)

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(provitamin A)-Converted in the body to vitamin A

Agricultural ApplicationsAgricultural Applicationsg ppg pp

Adoption of genetically modified (GM) Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about:because of questions about:

C f t f h ti-Crop safety for human consumption-Movement of genes into wild relatives-Loss of biodiversity

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y

Agricultural ApplicationsAgricultural ApplicationsBiopharming

-Transgenic plants are used to g pproduce pharmaceuticals

-Human serum albuminHuman serum albumin-Recombinant subunit vaccines

A i t N lk d bi -Against Norwalk and rabies viruses

-Recombinant monoclonal antibodies

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-Against tooth decay-causing bacteria

Transgenic MammalsTransgenic Mammals

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Medical ApplicationsMedical Applications

• The insertion of genetic material into human cells for the into human cells for the treatment of a disorder

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Recombinant DNA Recombinant DNA Vaccines?Vaccines?Vaccines?Vaccines?

Strategy for a subunit vaccine for herpes simplex

Gene TherapyGene Therapy

Treatment of SCID (severe combined immunodeficiency). SCID affects the

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( y)maturation of immune cells that develop in bone marrow. SCID sufferers lack the enzyme ADA (adenosine deaminase).