technique involving the insertion of a fragment of foreign dna into a vector capable of replicating...
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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
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