DNA Cloning

DNA Cloning• Isolation of DNA• Ligating the dna into a vector• Transformation of a host cell with the

recombinant dna • Selection of host cells harboring the

recombinant dna• Screening of cells for those harboring the

recombinant dna or producing the appropriate protein product

Cloning vectors

• Transport recombinant dna into a host cell

• a DNA molecule that carries foreign DNA into a host cell, replicates and produces many copies of itself and the foreign DNA

What cloning vectors must have

• 1. have an origin of replication• 2. be small• 3. have several unique restriction sites

• 4. Have selectable markers

Types of cloning vectors

• 1. bacterial vectorsGreatest variety of cloning vectors ( e. coli)

Plasmids- circular double strands that are extra chromosomal

• Functions:1. Encode substance s for

antibiotic resistance2. Bacteriocins3. Physiological functions4. Toxin- producing5. Virulence plasmids

Role of plasmids in dna cloning

• Engineered with unique restriction sites for insertion of foreign dna fragments

Types of cloning vectors

• 2. bacteriophage• A virus that infects a bacterium• Derived from a double strand genome that has a

single strand complementary ends of 12 nucleotides that can base pair which forms a circular dna molecule once inside the host cell

Types of cloning vectors

• 3. Cosmids• Engineered hybrids of phage dna

and plasmids• Plasmids with small portion of

bacteriophage vectors and replication origin for replication in bacterial hosts

• Used for cloning large dna fragments

Types of cloning vectors(for other organisms)

• 4. Yeast artificial chromosomes (YAC)• For eukaryotic molecular studies• Has the following necessary components:• A. centromere-distributes the chromosome to the daughter

cells• B. telomere- ensures that the end is correctly replicated and

protects against degradation• C. autonomously replicating sequence- specific dna that

enable the molecule to replicate

Types of cloning vectors(for other organisms)

• 5. Bacterial artificial chromosomes (bac)

• Synthetic vectors for large genome project and complex genomes

Types of cloning vectors(for other organisms)

• 6. Plant cloning vectors• Purposes:• A. generate resistance to disease, pests and herbicides• B. improve crop yield• C. quality and nutritional value• D. development of new ornamental plant characteristics• E. increase shelf life of common fruits and vegetables

• Commonly used plant vectors

• 1. tobacco mosaic virus• 2. Ti or tumor inducing plasmid- agrobacterium

tumafaciens

Types of cloning vectors(for other organisms)

• 7. Mammalian cell vectors• First eukaryotic infecting virus to be used

• Can infect the cells of several mammalian species including monkey cells

What is your stand on cloning?