Cloning and genetic Cloning and genetic engineeringengineering

bybyIvo FrébortIvo Frébort

CloningCloning

Clone: a collection of molecules or cells, all Clone: a collection of molecules or cells, all identical to an original molecule or cell identical to an original molecule or cell

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

bacteria 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

PlasmidsPlasmids

Naturally occurring extrachromosomal Naturally occurring extrachromosomal DNA DNA

Plasmids are circular dsDNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction Plasmids can be cleaved by restriction

enzymes, leaving sticky ends enzymes, leaving sticky ends Artificial plasmids can be constructed Artificial plasmids can be constructed

by linking new DNA fragments to the by linking new DNA fragments to the sticky ends of plasmid sticky ends of plasmid

Cloning VectorsCloning Vectors

Plasmids that can be modified to carry Plasmids that can be modified to carry new genes new genes

Plasmids useful as cloning vectors must havePlasmids useful as cloning vectors must have – aa replicator replicator (origin of replication) (origin of replication)

– a a selectable markerselectable marker (antibiotic resistance (antibiotic resistance gene)gene)

– a a cloning sitecloning site (site where insertion of (site where insertion of foreign DNA will not disrupt replication or foreign DNA will not disrupt replication or inactivate essential markersinactivate essential markers

DNA LibrariesDNA Libraries

Sets of cloned DNA fragments that together Sets of cloned DNA fragments that together represent the genes of a particular represent the genes of a particular

organismorganism Any particular gene may represent a tiny, Any particular gene may represent a tiny,

tiny fraction of the DNA in a given cell tiny fraction of the DNA in a given cell Can't isolate it directly Can't isolate it directly Trick is to find the fragment or fragments Trick is to find the fragment or fragments

in the library that contain the desired in the library that contain the desired genegene

Colony HybridizationColony Hybridization

Disk is treated with base or heated to Disk is treated with base or heated to convert dsDNA to ssDNA and convert dsDNA to ssDNA and incubated with probes incubated with probes

Colonies that bind probe hold the Colonies that bind probe hold the fragment of interestfragment of interest

Colony Hybridization

Southern BlotsSouthern Blots

The Polymerase Chain The Polymerase Chain ReactionReaction

What if you don't have enough DNA for What if you don't have enough DNA for colony hybridization or Southern blots?colony hybridization or Southern blots?

The small sample of DNA serves as The small sample of DNA serves as template for DNA polymerase template for DNA polymerase

Make complementary primers Make complementary primers Add primers in more than 1000-fold Add primers in more than 1000-fold

excess excess Heat to make ssDNA, then cool Heat to make ssDNA, then cool Run DNA polymerase (usually Run DNA polymerase (usually TaqTaq) ) Repeat heating, cooling, polymerase cycleRepeat heating, cooling, polymerase cycle

DNA sequencingDNA sequencingChemical Cleavage MethodChemical Cleavage Method

Four reactions are usedFour reactions are used GG-specific cleavage with -specific cleavage with dimethyl sulfatedimethyl sulfate, ,

followed by strand scission with piperidine followed by strand scission with piperidine G/AG/A cleavage: depurination with cleavage: depurination with mild acidmild acid, ,

followed by piperidine followed by piperidine C/TC/T cleavage: ring hydrolysis by cleavage: ring hydrolysis by hydrazinehydrazine, ,

followed by piperidine followed by piperidine CC cleavage: same method ( cleavage: same method (hydrazine and hydrazine and

piperidinepiperidine), but ), but high salthigh salt protects T protects T residuesresidues

DNA sequencingDNA sequencingChain Termination MethodChain Termination Method

Based on DNA polymerase reactionBased on DNA polymerase reaction Run four separate reactions Run four separate reactions Each reaction mixture contains dATP, Each reaction mixture contains dATP,

dGTP, dCTP and dTTP, one of which is dGTP, dCTP and dTTP, one of which is P-32-labelled P-32-labelled

Each reaction also contains a small Each reaction also contains a small amount of one amount of one dideoxynucleotidedideoxynucleotide: : either either ddddATP, ATP, ddddGTP, GTP, ddddCTP or CTP or ddddTTPTTP

Recombinant ProteinsRecombinant Proteins

Plant transformationPlant transformationCommon Strategies for Introducting Foreign Genes into Plant Cells:Common Strategies for Introducting Foreign Genes into Plant Cells: AgrobacteriumAgrobacterium infection infection Particle bombardmentParticle bombardment ElectroporationElectroporation

http://www.hort.purdue.edu/hort/courses/HORT250/animations/Leaf%20Disk%20Animation/leafdisk1.htmlhttp://www.hort.purdue.edu/hort/courses/HORT250/animations/Leaf%20Disk%20Animation/leafdisk1.html

Regeneration of transgenic plant from crown-gall callus

Tobacco plants overexpressing HvCKX genes

Transgenic AnimalsTransgenic Animals

Genes can be introduced into animals Genes can be introduced into animals by by transfectiontransfection - injection of plasmid - injection of plasmid DNA into recipient cellsDNA into recipient cells

Plasmids can be injected into fertilized Plasmids can be injected into fertilized eggs in miceeggs in mice

Expression is usually variable, because Expression is usually variable, because the gene is inserted randomlythe gene is inserted randomly

Growth hormone transfection produces Growth hormone transfection produces mice that are very large!mice that are very large!