TechniqueTechnique involving the insertion of a involving the insertion of a

fragment of fragment of foreign DNAforeign DNA into a into a vectorvector

capable of replicating autonomously in a capable of replicating autonomously in a

host cellhost cell (usually (usually Escherichia coliEscherichia coli). Growing ). Growing

the host cell allows the production of the host cell allows the production of

multiple copies of the inserted DNA for use multiple copies of the inserted DNA for use

in a in a variety of purposesvariety of purposes..

Foreign DNAForeign DNA

Host organismHost organism

Vector DNA for cloningVector DNA for cloning

Means of inserting foreign DNA into the vectorMeans of inserting foreign DNA into the vector

Method of placing the in vitro modified DNA Method of placing the in vitro modified DNA

into the host cellinto the host cell

Methods for selecting and/or screening cells Methods for selecting and/or screening cells

that that carry the inserted foreign DNAcarry the inserted foreign DNA

PolymerasesPolymerases

DNA Polymerase – catalyzes the polymerization of DNA Polymerase – catalyzes the polymerization of

deoxyribonucleotides along the template stranddeoxyribonucleotides along the template strand

DNA-dependent RNA PolymeraseDNA-dependent RNA Polymerase

Restriction EndonucleasesRestriction Endonucleases

NucleasesNucleases

Enzymes capable of cleaving the phosphodiester bonds Enzymes capable of cleaving the phosphodiester bonds

between nucleotide subunits of nucleic acidsbetween nucleotide subunits of nucleic acids

Other Modifying EnzymesOther Modifying Enzymes

LigasesLigases

forms phosphodiester bonds to join two pieces of forms phosphodiester bonds to join two pieces of DNADNA

utilizes ATP in the presence of Mgutilizes ATP in the presence of Mg++++

T4 DNA ligase for “blunt” endsT4 DNA ligase for “blunt” ends

KinasesKinases

transfers phosphate groups from donor moleculestransfers phosphate groups from donor molecules

phosphorylasephosphorylase

PhosphatasesPhosphatases

catalyzes the removal of 5’-phosphate residuescatalyzes the removal of 5’-phosphate residues

Foreign DNAForeign DNA

PCR productPCR product

genomic DNAgenomic DNA

complementary DNA (cDNA)complementary DNA (cDNA)

Host organismHost organism

bacterial host – bacterial host – E. coliE. coli

eukaryotic host – yeast (eukaryotic host – yeast (Saccharomyces cerevisiae)Saccharomyces cerevisiae)

other hosts – other yeasts, insect cells, etc.other hosts – other yeasts, insect cells, etc.

Vector DNAVector DNA

DNA molecule that functions as a “molecular carrier” DNA molecule that functions as a “molecular carrier”

that carry the DNA of interest into the host cell & facilitates that carry the DNA of interest into the host cell & facilitates

its replication.its replication.

PlasmidsPlasmids – used in cloning small segments of DNA (10-15 – used in cloning small segments of DNA (10-15

kb)kb)

CosmidsCosmids – plasmids containing DNA sequences ( – plasmids containing DNA sequences (coscos) from ) from

bacteriophage bacteriophage λλ used to clone larger used to clone larger

fragments fragments of up to 45 Kbof up to 45 Kb

Bacteriophage Bacteriophage λλ – used in cloning larger segments of DNA – used in cloning larger segments of DNA

(~20 kb)(~20 kb)

• small circular dsDNA that autonomously replicates small circular dsDNA that autonomously replicates

apart apart from the chromosome of the host cellfrom the chromosome of the host cell

• ““molecular parasites”molecular parasites”

• carry one or more genes some of which confer carry one or more genes some of which confer

resistance toresistance tocertain antibioticscertain antibiotics

• origin of replication (ORI) --- a region of DNA that origin of replication (ORI) --- a region of DNA that

allows multiplication of the plasmid within the allows multiplication of the plasmid within the

hosthost

• plasmid replication: stringent or relaxedplasmid replication: stringent or relaxed

small sizesmall size

known DNA sequenceknown DNA sequence

high copy numberhigh copy number

a selectable markera selectable marker

a second selectable genea second selectable gene

large number of unique restriction siteslarge number of unique restriction sites

Desirable properties of plasmids:Desirable properties of plasmids:

http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif

• viruses that infect bacteriaviruses that infect bacteria

• known dsDNA sequence of ~ 50 kbknown dsDNA sequence of ~ 50 kb

• linear double-stranded molecule with linear double-stranded molecule with

single-stranded complementary endssingle-stranded complementary ends

• cohesive termini (cos region)cohesive termini (cos region)

• can accept large pieces of foreign DNAcan accept large pieces of foreign DNA

• tremendous improvement over the tremendous improvement over the

yearsyears

• can be reconstituted can be reconstituted in vitroin vitro

Desirable properties of Desirable properties of λλ

phage:phage:

• modified plasmids containing modified plasmids containing coscos sequences sequences

• carry an ORI & an antibiotic resistance markercarry an ORI & an antibiotic resistance marker

• can accommodate ~35 to 45 kb of foreign DNAcan accommodate ~35 to 45 kb of foreign DNA

• can be propagated as plasmidscan be propagated as plasmids

• can be introduced into host by standard can be introduced into host by standard

proceduresprocedures

• chief technical problems occur when used for chief technical problems occur when used for

library constructionlibrary construction

Means of inserting foreign DNA into the vectorMeans of inserting foreign DNA into the vector

LigationLigation of the DNA into the linearized vector of the DNA into the linearized vector

• two or more fragments of DNA (blunt/cohesive)two or more fragments of DNA (blunt/cohesive)

• buffer containing ATPbuffer containing ATP

• T4 DNA ligaseT4 DNA ligase

Requirements for a ligation reaction:Requirements for a ligation reaction:http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif

Method of placing the Method of placing the in vitroin vitro modified DNA modified DNA

into the host cellinto the host cell

TransformationTransformation into the host cell into the host cell

• bacterial cells take up naked DNA moleculesbacterial cells take up naked DNA molecules

• cells are made “competent”cells are made “competent”

• cells treated with ice-cold CaClcells treated with ice-cold CaCl22 then heat-shocked then heat-shocked

• efficiency of 10efficiency of 1077 to 10 to 1088 transformed colonies/ transformed colonies/μμg DNAg DNA

• maximum transformation frequency of 10maximum transformation frequency of 10-3-3

ElectroporationElectroporation of the DNA into the host cell of the DNA into the host cell

• “ “electric field-mediated electric field-mediated

membrane membrane permeabilization” permeabilization”

• high strength electric field in the high strength electric field in the

presence of DNApresence of DNA

• protocols differ for various speciesprotocols differ for various species

• efficiencies of 10efficiencies of 1099 per per μμg DNA (3 g DNA (3

kb) kb) & 10& 1066 (136 kb) (136 kb)

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TransfectionTransfection of the DNA of the DNA

• DNA is packaged DNA is packaged in vitroin vitro into phage particlesinto phage particles

• phages are allowed to infect bacterial cellsphages are allowed to infect bacterial cells

• term also used in DNA transfer to eukaryotic cellsterm also used in DNA transfer to eukaryotic cells

• DNA is transiently expressedDNA is transiently expressed

ConjugationConjugation

• natural transmission from donor to recipientnatural transmission from donor to recipient

• host cell that is not readily transformedhost cell that is not readily transformed

• form cell to cell junctionsform cell to cell junctions

Methods for selecting and/or screening cells Methods for selecting and/or screening cells

that that carry the inserted foreign DNAcarry the inserted foreign DNA

SelectionSelection refers to application of conditions that refers to application of conditions that

favors the growth of cells or phages that favors the growth of cells or phages that

carry carry the vector or vector and the vector or vector and insert.insert.

• antibiotic resistanceantibiotic resistance

• nutrient requirementsnutrient requirements

• plaque formationplaque formation

ScreeningScreening allows all cells to grow, but tests the allows all cells to grow, but tests the

resulting clones for the presence of the insert resulting clones for the presence of the insert in in

the vector.the vector.

• antibiotic resistance/sensitivityantibiotic resistance/sensitivity

• nutrient requirementsnutrient requirements

• plaque typeplaque type

• blue-white selection (blue-white selection (ββ-galactosidase)-galactosidase)

• specific (hybridization, antibodies, PCR)specific (hybridization, antibodies, PCR)

http://www.eppendorfna.com/applications/images/gel_cleanup1.jpg

• LacZ gene – encodes for LacZ gene – encodes for ββ-galactosidase-galactosidase

• X-Gal – substrate for the enzymeX-Gal – substrate for the enzyme

• IPTG (isopropyl-[beta]-D-thiogalactopyranoside)IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer– inducer

• cloning sites within the LacZ genecloning sites within the LacZ gene

• disruption of gene by insertiondisruption of gene by insertion

of the foreign DNAof the foreign DNA

• blue – functional proteinblue – functional protein

• white – non-functional proteinwhite – non-functional protein

DNA isolation for:DNA isolation for:

making probesmaking probes

restriction mappingrestriction mapping

sequencingsequencing

reintroduction into organismreintroduction into organism

Establishment of collections: DNA LibrariesEstablishment of collections: DNA Libraries

Further molecular studies: production of special Further molecular studies: production of special

proteinsproteins

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http://www.bio.indiana.edu/~chenlab/potocols/MolecularClonging.htm

http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html

http://www.protocol-online.org/forums/index.php?showforum=30

http://bioresearch.ac.uk/browse/mesh/D003001.html

http://www.jax.org/~jcs/techniques/techniques.html

http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/clones.html

http://opbs.okstate.edu/~Melcher/MG/MGW4/MG428.html http://www-micro.msb.le.ac.uk/109/GeneticEngineering.html

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/cloningvectors.html