Cloning vectors

The challenge of inserting new DNA into a cell lies not just in getting it into the host cell, but in getting it to replicate in the host cell as it divides. DNA polymerase, the enzyme that catalyzes replication, does not bind to just any sequence of DNA to begin replication. But  it recognizes a specific sequence, the origin of replication If the new DNA is to be replicated, it must become part of a segment of DNA that contains an origin of replication, called a replicon, or replication unit.
There are two general ways in which the newly introduced DNA can become part of a replication unit.
1.It can be inserted into a host chromosome after entering the host cell. Although this insertion is often a random event, it is nevertheless a common method of integrating a new gene into a
host cell.
2. The new DNA can enter the host celi as part of a carrier DNA sequence or the vector that already
has the appropriate origin of replication.
A vector should have four characteristics:

1. The ability to replicate independently in the host cell
2. Arecognition sequence for a restriction enzyme, allowing the vector to be cut and combined with the new DNA
3. A reporter gene that will announce its presence in the host cell
4. A small size in comparison to the host chromosomes for ease of isolation.

Some common vectors are

 Plasmids

Plasmids  are naturally occurring bacterial chromosomes, have all four of the properties needed for a useful vector. First, they are small  for eg.an E. coli plasmid has 2,000–6,000 base pairs, as compared with the main E. coli chromosome, which has more than 3 million base pairs. Being small, a plasmid often has only a single recognition site for a given restriction enzyme  This property is essential because it allows for the

insertion of new DNA at only one location.. When the plasmid is cut with a restriction enzyme, it is transformed into a linear molecule with sticky ends. The sticky ends of another DNA fragment cut with the same restriction enzyme can pair with the sticky ends of the plasmid, resulting in a circular plasmid containing the new DNA.

Many plasmids contain genes that confer resistance to antibiotics. This property provides a genetic marker for host cells carrying the recombinant plasmid.Plasmids have an origin of replication and can replicate independently of the host chromosome. It is not uncommon for a bacterial cell with a single main chromosome to contain hundreds of copies of a recombinant plasmid.Many of the plasmids have a single

marker for antibiotic resistance.

Viruses 

 Constraints on plasmid replication limit the size of the new DNA that can be inserted into a plasmid to about 10,000 base pairs. Some prokaryotic genes may be small and is about 10,000 base pairs is much smaller than most eukaryotic genes with their introns and extensive flanking sequences. A vector that accommodates

larger DNA inserts is needed.Both prokaryotic and eukaryotic viruses are often used as vectors for eukaryotic DNA. Bacteriophage λ, which infectsE. coli, has a DNA genome of about 45,000 base pairs. If the genes that cause the host cell to die and lyse,about 20,000 base pair,are eliminated, the virus can still attach to a host cell and inject its DNA. The deleted 20,000 base pairs can be replaced with DNA from another organism, thereby creating highly usable recombinant viral DNA. Because viruses infect cells naturally, they offer a great advantage as vectors over plasmids, which often require artificial means to coax them to enter cells.

Artificial  chromosomes

Bacterial plasmids are not good vectors for yeast hosts because prokaryotic and eukaryotic DNA sequences use different origins of replication. Thus a recombinant bacterial plasmid will not replicate in yeast. To solve  this problem, scientists have created a “minimalist chromosome” called the yeast artificial chromosome, or YAC . This artificial DNA molecule contains not only the yeast origin of replication, but the yeast centromere and telomere sequences as well, making it a true eukaryotic chromosome. YACs also contain artificially synthesized restriction sites and useful reporter genes (for yeast nutritional requirements). YACs

are only about 10,000 base pairs in size, but can accommodate 50,000 to 1.5 million base pairs of inserted DNA. These artificial chromosomes carry out eukaryotic DNA replication and gene expression normally in yeast cells.

Plasmid vectors for plants:

 An important vector for carrying  new DNA into many types of plants is a plasmid that is found in Agrobacterium tumefaciens. This bacterium lives in the soil and causes a plant disease called crown gall,

which is characterized by the presence of growths, or tumors, in the plant. A. tumefaciens contains a plasmid called Ti (for tumor-inducing) . The Ti plasmid contains a transposon, called T DNA, that inserts copies of itself into the chromosomes of infected plant cells. The T DNA contains recognition sequences for restriction enzymes, so that new DNA can be inserted into it. When the T DNA is thus altered, the plasmid no longer produces tumors, but the transposon, with the new DNA, can still be inserted into the host cell’s chromosomes. A plant cell containing this DNA can then be grown in culture or induced to form a new, transgenic plant.