DNA Cloning and Vectors

• DNA cloning introduces foreign DNA sequences into a vector.
• Vectors are artificially constructed DNA molecules that allow for replication of foreign DNA within a host cell, like bacteria or yeast.
• Chromosomes are too large to clone whole, so genomic DNA must be fragmented.
• Restriction enzymes can cut DNA.

Requirements for DNA Cloning

• Cloning vectors contain signals for DNA propagation, origin of replication, and a selectable marker.
• Selectable markers distinguish transformed and non-transformed cells.
• DNA modifying enzymes are used to cut and re-ligate the vector DNA to create recombinant DNA.
• Host organism is needed, it accepts the engineered DNA (transformable) and propagates it without rearrangement (recombination deficient).

Cloning Vectors

• Cloning vectors are small DNA pieces from viruses or plasmids, stably maintained in an organism, and accept foreign DNA fragments during cloning.
• Vectors enable convenient insertion/removal of DNA fragments through restriction enzymes that create overhangs that can then be ligated together.
• After a DNA fragment is cloned into a cloning vector, it can be subcloned into another vector for specific uses.
• Cloning vectors are usually genetically engineered plasmids.
• Cloning is typically done using Escherichia coli (E. coli).
• Cloning vectors in E. coli are plasmids, bacteriophages (like phage λ), cosmids, and bacterial artificial chromosomes (BACs).
• DNA that cannot be stably maintained in E. coli can be used in other organisms like yeast.
• Cloning vectors in yeast include yeast artificial chromosomes (YACs).
• Different cloning vectors are used for different cloning experiments.
• Vectors are chosen based on the size and type of DNA to be cloned
• insert size
• vector size
• restriction sites
• copy number
• cloning efficiency
• ability to screen for inserts
• Cloning vectors have multiple cloning sites (collections of different restriction sites), an appropriate origin of replication, and a selectable marker.
• Common vectors include plasmids, cosmids, YACs, and BACs, each with its own advantages and disadvantages.
• Replication origin is a DNA segment recognized by cellular DNA-replication enzymes.
• Without a replication origin, DNA cannot replicate in the cell.
• Many cloning vectors have a multiple cloning site or polylinker (a DNA segment with unique sites for restriction endonucleases located next to each other).
• Restriction sites in the polylinker are not found elsewhere in the plasmid.
• Cutting plasmids with a restriction enzyme that recognizes a site in the polylinker does not disrupt essential vector features.

Plasmid Vectors

• Plasmid vectors are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules.
• Advantages:
• Small, easy to handle
• Useful for cloning small DNA fragments (< 10kbp).
• Disadvantages:
• Less useful for cloning large DNA fragments (> 10kbp).

Cutting DNA

• Restriction endonucleases (restriction enzymes) create sticky or blunt ends of DNA.
• Nomenclature for naming restriction enzymes
• EcoRI
• E = genus (Escherichia)
• co = species (coli)
• R = strain
• I = # of enzyme

Pasting DNA

• Uses complementary ends (sticky ends) via H-bonding.
• Ligase forms phosphodiester bonds to seal strands together.

DNA Cloning Steps

• Isolate DNA from an organism.
• Cut the DNA into pieces with a restriction enzyme.
• Restriction enzymes recognize and cut specific DNA sequences.
• Insert (ligate) each piece individually into a cloning vector that has been cut with the same restriction enzyme, which creates a recombinant DNA molecule.
• Recombinant DNA molecules are constructed in vitro and contain sequences from two or more distinct DNA molecules.
• Introduce recombinant DNA molecules into a host such as E. coli.
• Replication of the recombinant DNA molecule happens occurs in the host cell during molecular cloning, producing many identical copies called clones.
• As the host organism reproduces, the recombinant DNA molecules are passed on to all progeny, resulting in a population of cells carrying the cloned sequences.

Expression Vectors

• An expression vector is a cloning vector with the necessary regulatory sequences to allow transcription and translation of a cloned gene or genes.
• Expression vectors are used to produce the protein encoded by a cloned gene in the transformed host.
• Expression vectors create pharmaceutically active proteins by the biotechnology industry with an appropriate host.
• Expression vectors are derivatives of plasmid cloning vectors used in the same host.
• Additions to the features of an E. coli cloning vector:
• Strong promoter (upstream of multiple cloning site).
• Transcription terminator (downstream of the multiple cloning site).
• DNA sequence encoding the Shine-Dalgarno sequence for translation initiation (located between the promoter and multiple cloning site).
• A ribosome binding site near an initiating ATG codon.
• Cloning occurs in an expression vector
• Expression vectors produce large amounts of specific proteins.
• Permits structural and functional studies of proteins.
• Expression vectors are useful when proteins are rare cellular components or difficult to isolate.

Promoters

• A promoter is a region of DNA that initiates transcription of a particular gene.
• Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA.