Cloning vector

A cloning vector is a small piece of DNA used as a carrier. It is designed to be stably maintained within a living organism. Scientists insert a foreign DNA fragment into the vector to make many copies of that fragment. This process is known as cloning.

Vectors can be derived from various sources. They might come from a virus, a higher organism's cell, or a bacterium's plasmid. These tools are essential for molecular biology research.

To insert DNA, scientists often use a specific mechanism involving enzymes. First, a restriction enzyme acts like molecular scissors to cut the DNA. This cutting creates ends that are either blunt or have overhangs called sticky ends. If the vector and the foreign DNA have compatible ends, they can be joined together. This joining process is called molecular ligation.

Other methods exist for faster work. In TOPO cloning, an enzyme called topoisomerase I is attached to the vector ends. This "TOPO-activated" vector can accept a PCR product and form a circular vector quickly. Another method, the Gateway cloning system, uses DNA recombination instead of cutting and joining.

Every functional cloning vector requires certain key features. A suitable cloning site is necessary to allow DNA to be inserted or removed. Many vectors use a multiple cloning site (MCS), also called a polylinker. This area contains many unique restriction sites for easy manipulation. Another vital feature is the origin of replication, often called an "ori." This allows the vector to replicate itself within the host. Some vectors are called shuttle vectors because they can be maintained in two different organisms.

To identify successful cloning, vectors use a selectable marker. This marker helps scientists select only the cells that have taken up the vector. A common marker is an antibiotic resistance gene. For example, the beta-lactamase gene allows a cell to resist ampicillin. Some plasmids, like pACYC177, carry two markers, such as ampicillin and kanamycin resistance. Other vectors use auxotrophic selection markers. These allow organisms like yeast to grow in minimal medium using genes like LEU2 or URA3.

Some vectors even use lethal genes to ensure only successful clones survive.

Reporter genes are another tool used to facilitate screening. These genes help scientists visually identify successful clones. One common method is blue-white selection using the lacZα fragment. This can be seen on agar plates where colonies change color. Other reporter genes include green fluorescent protein (GFP) or luciferase. These can make the successful clones glow. Some vectors are also designed as expression vectors. These contain a promoter and a ribosomal binding site (RBS) to produce proteins from the cloned gene.

Scientists choose specific vectors based on the size of the DNA fragment. Plasmids are the most common standard vectors. They can usually carry inserts up to 15 kb in size. Some plasmids, like the pUC19, have a high copy number. This means they can have 500 to 700 copies per cell, which provides a high yield. However, if a protein is toxic, a low-copy-number plasmid might be better. For larger fragments, scientists use bacteriophages like λ phage. These can carry between 5 kb and 24 kb depending on the vector type.

Even larger DNA fragments require specialized vectors. Cosmids are used for fragments between 28 and 45 kb. Bacterial artificial chromosomes (BACs) can carry up to 350 kb. These are often maintained in E. coli with only one copy per cell. For extremely large tasks, such as mapping genomes in the Human Genome Project, yeast artificial chromosomes (YACs) are used. These can carry more than 1 megabase of DNA. Finally, human artificial chromosomes may be used for gene delivery into human cells without the risks of viral vectors.