Lecture # 04Lecture # 04

Cloning Vectors Cloning Vectors

Recombinant DNA Recombinant DNA TechnologyTechnology

Common General Cloning Strategy• Target DNA from donor organism extracted, cut with compatible

restriction endonuclease and ligated into a cloning vector.

• Recombinant construct transferred into host cell.

• Host cells which do not take up construct are eliminated by selection protocol

• Host cell library screened to identify desired clone.

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transformation

RestrictionEndonuclease

DNA Ligase

Cloning VectorsCloning Vectors

The molecular analysis of DNA has been made The molecular analysis of DNA has been made possible by the cloning of DNA. possible by the cloning of DNA.

The two molecules that are required for cloning The two molecules that are required for cloning are the are the DNA to be cloned DNA to be cloned and and a cloning vectora cloning vector. .

Cloning vectorCloning vector - a DNA molecule that - a DNA molecule that carries carries foreign DNA foreign DNA into a host cell, into a host cell, replicatesreplicates inside a inside a host (bacterial or yeast) cell and produces host (bacterial or yeast) cell and produces many copiesmany copies of itself and the foreign DNA. of itself and the foreign DNA.

Essential features of all Essential features of all cloning vectorscloning vectors

• Ideally they should be fairly small DNA molecule.Ideally they should be fairly small DNA molecule.(to facilitate isolation and handling)(to facilitate isolation and handling)

• Origen of replication: Origen of replication: sequences that permit the sequences that permit the propagation of itself in host cell.propagation of itself in host cell.

• A A cloning site to insert foreign DNAcloning site to insert foreign DNA; The vectors ; The vectors contain a site that can be cut by unique contain a site that can be cut by unique restriction restriction enzymes. enzymes.

• Some sort of Some sort of selectable markers: selectable markers: to select host cell to select host cell containing a vector with foreign DNA; containing a vector with foreign DNA; usually accomplished usually accomplished by selectable markers for drug resistance by selectable markers for drug resistance

Types of Cloning Types of Cloning VectorsVectors

PlasmidPlasmid - an extrachromosomal circular DNA molecule that autonomously - an extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or replicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or 0.1-10 kilobases (kb) 0.1-10 kilobases (kb)

PhagePhage - derivatives of bacteriophage lambda; linear DNA molecules, - derivatives of bacteriophage lambda; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb cycle; cloning limit: 8-20 kb

CosmidsCosmids - - is a combination is a combination of plasmids and phage; cloning limit - 35-50 kb of plasmids and phage; cloning limit - 35-50 kb

Bacterial Artificial Chromosomes (BAC)Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-F - based on bacterial mini-F plasmids. cloning limit: 150-300 kb plasmids. cloning limit: 150-300 kb (350kb in some books).(350kb in some books).

Yeast Artificial Chromosomes (YACYeast Artificial Chromosomes (YAC)) - an artificial chromosome that - an artificial chromosome that contains telomeres, origin of replication, a yeast centromere, and a contains telomeres, origin of replication, a yeast centromere, and a selectable marker for identification in yeast cells; cloning limit: 100-2000 kb selectable marker for identification in yeast cells; cloning limit: 100-2000 kb (3000kb in some books)(3000kb in some books)

General Steps of Cloning with Any General Steps of Cloning with Any VectorVector

prepare the vector and DNA to be cloned by digestion prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary with restriction enzymes to generate complementary ends. ends.

ligate the foreign DNA into the vector with the enzyme ligate the foreign DNA into the vector with the enzyme DNA ligase DNA ligase

introduce the DNA into bacterial cells (or yeast cells for introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation YACs) by transformation

select cells containing foreign DNA by screening for select cells containing foreign DNA by screening for selectable markers (usually drug resistance) selectable markers (usually drug resistance)

Cloning ToolsCloning Tools

Restriction endonucleasesRestriction endonucleases LigaseLigase VectorsVectors HostHost Methods for introducing DNA into a host cellMethods for introducing DNA into a host cell

Cutting DNACutting DNA

Restriction endonucleases Restriction endonucleases (restriction enzymes)(restriction enzymes)• sticky endssticky ends• blunt endsblunt ends

NomenclatureNomenclature• EcoEcoRIRI• EE = genus ( = genus (EscherichiaEscherichia))• coco = species ( = species (colicoli))• R = strainR = strain• I = # of enzymeI = # of enzyme

Blunt & Sticky ends

Pasting DNAPasting DNA

Complementary ends Complementary ends (sticky ends) H-bond(sticky ends) H-bond

Ligase forms Ligase forms phosphodiester bond phosphodiester bond to seal strands to seal strands together.together.

What determines the choice What determines the choice vector?vector?

insert sizeinsert size

vector sizevector size

restriction sitesrestriction sites

copy numbercopy number

cloning efficiencycloning efficiency

ability to screen for insertsability to screen for inserts

How to clone DNA How to clone DNA (Summary)(Summary)

Isolation of cloning vector Isolation of cloning vector (bacterial plasmid) & gene-(bacterial plasmid) & gene-source DNA (gene of interest)source DNA (gene of interest)

Insertion of gene-source DNA Insertion of gene-source DNA into the cloning vector using into the cloning vector using the same restriction enzyme; the same restriction enzyme; bind the fragmented DNA with bind the fragmented DNA with DNA ligaseDNA ligase

Introduction of cloning vector Introduction of cloning vector into cells (transformation by into cells (transformation by bacterial cells)bacterial cells)

Cloning of cells (and foreign Cloning of cells (and foreign genes)genes)

Identification of cell clones Identification of cell clones carrying the gene of interestcarrying the gene of interest

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