DNA Recombinant Technology DNA Recombinant Technology

DNA recombinantDNA recombinant

Genetic Engineering

The manipulation of an organism endowment by introducing or eliminating specific gene

A gene of interest is inserted into another organism, enabling it to be cloned, and thus studied more effectively

Design and construction of new combinations of genes (DNA)

New combinations/arrangements of DNA

DNA cloning

Technology used in the isolation or synthesis and joining together of unlike pieces of DNA

DNA Recombinant Technology DNA Recombinant Technology

These recombinant DNA molecules can then be introduced into bacteria, yeasts, or other cells where they can replicate and

function (code for protein synthesis)

The Application of DNA Recombinant The Application of DNA Recombinant Technology Technology

Overview of Genetic Engineering

Gene of interest is isolated from appropriate organism Gene is recombined with a vector (carrier) DNA molecule Recombinant DNA is introduced into appropriate host cell Recombinant DNA is expressed at high levels in host cell

Gene product may be purified for use in treatments (antibiotics, hormones, etc.)

WhyWhy

Detailed studies of the structure and function of a Detailed studies of the structure and function of a gene at the molecular level require large quantities gene at the molecular level require large quantities

of the individual gene in pure formof the individual gene in pure form

Cloning

A collection of molecules or cells, all A collection of molecules or cells, all identical to an original molecule or cellidentical to an original molecule or cell

To "clone a gene" is to make many copies of it To "clone a gene" is to make many copies of it - for example, in a population of bacteria - for example, in a population of bacteria

Gene can be an exact copy of a natural gene Gene can be an exact copy of a natural gene Gene can be an altered version of a natural Gene can be an altered version of a natural

gene gene Recombinant DNA technology makes it Recombinant DNA technology makes it

possible possible

ToolsTools

VectorRestriction and ligation enzymes

Host Cells

VectorVector Carriers move DNA from test tubes back into Carriers move DNA from test tubes back into

cellscells Pieces of DNA that can accept, carry, and Pieces of DNA that can accept, carry, and

replicate other pieces of DNA replicate other pieces of DNA An autonomously replicating genetic element An autonomously replicating genetic element

used to carry DNA fragments into a host for used to carry DNA fragments into a host for the purpose of gene cloningthe purpose of gene cloning

1. Bacterial plasmids

2. Bacteriophages (lambda phage)

3. Viruses4. Yeast cells

Cloning vectorsVector system Host cell Insert capacity (kb)

Plasmid E. coli 0.1-10

Bacteriophage l E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (bacterial artificial chromosome)

E. coli 50-300

P1 bacteriophage-derived AC

E. coli 100-300

YAC Yeast 100-2,000

Human AC Cultured human cells

>2,000

PlasmidsPlasmidsNaturally occurring extra-chromosomal

DNA

Plasmids are circular double stranded DNA Plasmids can be cleaved by restriction

enzymes, leaving sticky ends Artificial plasmids can be constructed by

linking new DNA fragments to the sticky ends of plasmid

Maximum size of insert is about 10 kb.

LambdaLambda It has a genome of about 50 kb of linear

DNA Only 37 to 52 kb DNA fragments can be

packaged into the lambda head. Insertion vectors can hold up to 7 kb of

cDNA. Its life cycle is conducive to the use as a

cloning vector The lytic cycle can be supported by only a

portion of the genes found in the lambda genome.

Lambda life cycle.

The lytic life cycle produces phage particles immediately

The lysogenic life cycle requires genes in the middle of the genome, which can be replaced

Lambda genome

Cosmid vectorsCosmid vectors

Hybrid between a lambda vector and a plasmid. It can contain 33 to 45 kb.

Bacterial Artificial Bacterial Artificial chromosomes (BAC) vectorschromosomes (BAC) vectors

These vectors are based on the E. coli F factorThese vectors are maintained at 1-2 copies per cell and can hold > 300 kb of insert DNA.

Problems are low DNA yield from host cells.

Bacteriophage P1 These vectors are like lambda and can These vectors are like lambda and can

hold up to 110 to 115 kb of DNA . hold up to 110 to 115 kb of DNA . This DNA can then be packaged by the This DNA can then be packaged by the

P1 phage protein coat.P1 phage protein coat.The use of T4 in vitro packaging systems The use of T4 in vitro packaging systems

can enable the recovery of 122 kb insertscan enable the recovery of 122 kb inserts

Yeast Artificial Chromosomes

Many DNA fragments cannot be propagated in bacterial cells.

Therefore yeast artificial chromosomes can be built with a few specific

components.1.Centromere

2.Telomere3.Autonomously replicating sequence (ARS)

Genomic DNA is ligated between two telomeres and the ligation products are transformed into yeast cells

YAC cloning system

Cloning VectorsCloning Vectors

Plasmids that can be modified to carry new genes

Plasmids useful as cloning vectors must have Plasmids useful as cloning vectors must have • a replicator (origin of replication) a replicator (origin of replication) • a selectable marker (antibiotic resistance a selectable marker (antibiotic resistance

gene) gene) • a cloning site (site where insertion of foreign a cloning site (site where insertion of foreign

DNA will not disrupt replication or inactivate DNA will not disrupt replication or inactivate essential markersessential markers

Vectors

Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker

Coli Plasmid

pBR322pBR322

The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid pMB1); (2) rop gene coding for the

Rop protein, which promotes conversion of the unstable RNA I – RNA II complex to a stable complex and serves to decrease copy number (source –

plasmid pMB1); (3) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4) tet gene, encoding tetracycline

resistance protein (source – plasmid pSC101).

pUC18/19pUC18/19

pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids, 2686 bp in length. They are identical except that they contain multiple cloning sites (MCS) arranged in opposite

orientations. pUC18/19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid (source – plasmid pBR322). The high copy number of pUC plasmids is a

result of the lack of the rop gene and a single point mutation in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lacZ gene encoding the N-terminal fragment of beta-

galactosidase (source – M13mp18/19). This fragment, whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa) complementation with a defective form of beta-galactosidase

encoded by host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize both fragments of the enzyme and form blue colonies on media with X-Gal. Insertion of DNA into

the MCS located within the lacZ gene (codons 6-7 of lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-galactosidase and abolishes alfa-complementation. Bacteria

carrying recombinant plasmids therefore give rise to white colonies.

Agrobacterium tumefaciensAgrobacterium tumefaciens

Genetic structure of the Octopine Ti plasmid

TL TRAux Cyt Opines

Oncogenes

Fig. 3

Binary vector system

Binary vector system

A typical plasmid vector with a A typical plasmid vector with a polylinkerpolylinker

Chimeric PlasmidsChimeric Plasmids

Named for mythological beasts with body parts from several creatures

After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted

Ends of the plasmid/fragment are closed to form a "recombinant plasmid"

Plasmid can replicate when placed in a suitable bacterial host

Directional CloningDirectional Cloning

Often one desires to insert foreign DNA in a particular orientation

This can be done by making two

cleavages with two different restriction enzymes

Construct foreign DNA with same two restriction enzymes

Foreign DNA can only be inserted in one direction

Host Cells

Propagation of a DNA sequence must take place inside a living cell (host cells)

Eschericia coli:It provides a relatively simple and well understood

genetic environmentThe way to isolate plasmid is understood

It contains a single chromosome of approximately 5 Mbp

The genetic code is nearly universalIt replicates once every 22 minutes

It grows best with incubation at 37°Cin a culture medium that approximately the nutrient available in the

human digestive tract

Bacterial transformation

The cellular uptake and expression of DNA in a bacteria

Introduction of DNA into competent cell of bacteriaRequested element in

transformation:1. A suitable host organism in which to

insert the gene2. A self-replicating

vector to carry the gene into the host

organism3. A means of selection

for host cells that have taken up the gene

Selection of Transformant

A particularly important selective advantage offered by plasmid is antibiotic resistance gene

It encodes for proteins that disable antibiotics secreted by microorganism with which bacteria

competeAntibiotics function by several different

mechanism

Antibiotics resistance:A selectable marker that allows one to positively

identify cells that have been induced to take up plasmid DNA

Penicillin family (including ampicillin) interfere with cell

wall biosynthesisKanamycin, tetracyclin, and chloramphenicol arrest

bacterial cell growth by blocking various steps in protein synthesis

Selectable Marker Gene

Antibiotic Description

Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by b-lactamase, which cleaves the b-lactam ring of amp

Kanamycin (Kan) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase

Neomycin (Neo) Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase

Tetracycline (Tet) Binds to 30S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell

Protein expression

- Gene is inserted into plasmid- Plasmid is transformed into a host cell (E. coli)- Cell culture is prepared- Each cell contains several copies of the plasmid with gene

- Gene expression leads to the production of protein

- Protein level may reach 30% of

total cellular protein-Isolation of protein

Restriction EnzymesRestriction Enzymes

Molecular scissors which isolated from bacteria where they are used as Bacterial defense against viruses

Molecular scalpels to cut DNA in a precise and predictable manner

Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction

sites, and then cleave both DNA strands at this site

A class of endo-nucleases that cleavage DNA after recognizing a specific sequence

Members of the class of nucleases

Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and

RNA molecules

EndonucleaseCleave nucleic acids at internal position

ExonucleaseProgressively digest from the ends of the nucleic acid molecules

Nuclease

Endonuclease

Type Characteristics

I Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from

recognition site ATP is required

II It has only restriction site activity Its cut is predictable and consistent manner at a

site within or adjacent to restriction site It require only magnesium ion as cofactor

III Have both restriction and modification activityCut at sites closed to recognition site ATP is required

There are already more than 1200 type II enzymes isolated from prokaryotic organism

They recognize more than 130 different nucleotide sequence

They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence

Palindromic sequence:The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand

To Avoid confusion, restriction endo-nucleases are named according to the following nomenclature

Restriction Enzymes

The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated

The second and third letters are usually the initial letters of the organisms species name. It is written in italic

A fourth letter, if any, indicates a particular strain organism

Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

Nomenclature

NomenclatureEcoEcoRIRI E : Genus EscherichiaE : Genus Escherichia

co: Species colico: Species coli

R : Strain RY13R : Strain RY13

I : First endonuclease isolatedI : First endonuclease isolated

BamBamHIHI B : Genus BacillusB : Genus Bacillus

am: species amyloliquefaciensam: species amyloliquefaciens

H : Strain HH : Strain H

I : First endonuclease isolatedI : First endonuclease isolated

HinHindIIIdIII H : Genus HaemophilusH : Genus Haemophilus

in : species influenzaein : species influenzae

d : strain Rdd : strain Rd

III : Third endonuclease isolatedIII : Third endonuclease isolated

SpecificityEnzymeEnzyme SourceSource SequenceSequence EndEnd

BamHIBamHI Bacillus Bacillus amyloliquefaciens Hamyloliquefaciens H

GGGATCCGATCC StickStickyy

BglIIBglII Bacillus globigiiBacillus globigii AAGATCTGATCT StickStickyy

EcoRIEcoRI Escherichia coli RY13Escherichia coli RY13 GGAATTCAATTC StickStickyy

EcoRIIEcoRII Escherichia coli R245Escherichia coli R245 CCTGGCCTGG StickStickyy

HaeIIIHaeIII Haemophilus aegyptiusHaemophilus aegyptius GGGGCCCC BluntBlunt

HindIIHindII Haemophilus influenzae Haemophilus influenzae RdRd

GTPyGTPyPuACPuAC BluntBlunt

HindIIIHindIII Haemophilus influenzae Haemophilus influenzae RdRd

AAAGCTTAGCTT StickStickyy

HpaIIHpaII Haemophilus Haemophilus parainfluenzaeparainfluenzae

CCCGGCGG StickStickyy

NotINotI Nocardia otitidis-Nocardia otitidis-caviarumcaviarum

GCGCGGCCGGGCCGCC

StickStickyy

PstIPstI Providencia stuartii 164Providencia stuartii 164 CTGCACTGCAGG StickStickyy

Restriction Product

Restriction enzymesRestriction enzymes

degenerate or specific sequences

kind of ends produced (5’ or 3’ overhang (cohesive=sticky), blunt=flush)

number of nucleotides recognized (4, 6,8 base-cutters most common)

whether cleavage occurs within the recognition sequence

Restriction enzymes can be grouped by:

A restriction enzyme (A restriction enzyme (EcoEcoRI)RI)

1. 6-base cutter

4. produces a 5’ overhang (sticky end)

2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme)

Restriction enzymesRestriction enzymes