Joining DNA molecules

Once the proper DNA fragments have been obtained, they must be joined. When cleavage with a restriction endonuclease creates cohesive ends, these can be annealed with a similarly cleaved DNA from another source, including a vector molecule. When such molecules associate, the joint has nicks a few base pairs apart in opposite strands. The enzyme DNA ligase can then repair these nicks to form an intact, duplex recombinant molecule, which can be used for transformation and the subsequent selection of cells containing the recombinant molecule. Cohesive ends can also be created by the addition of synthetic DNA linkers to blunt-ended DNA molecules.

Another method for joining DNA molecules involves the addition of homopolymer extensions to different DNA populations followed by an annealing of complementary homopolymer sequences. For example, short nucleotide sequences of pure adenine can be added to the 3′ ends of one population of DNA molecules and short thymine blocks to the 3′ ends of another population. The two types of molecules can then anneal to form mixed dimeric circles that can be used directly for transformation.

The enzyme T4 DNA ligase carries out the intermolecular joining of DNA substrates at completely base-paired ends; such blunt ends can be produced by cleavage with a restriction enzyme or by mechanical shearing followed by enzyme treatment.

Transformation
The desired DNA sequence, once attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is usually referred to as transferring.

Transformation in any organism involves (1) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity. See also Transformation (bacteria).

Escherichia coli is usually the host of choice for cloning experiments, and transformation of E. coli is an essential step in these experiments. Escherichia coli treated with calcium chloride are able to take up DNA from bacteriophage lambda as well as plasmid DNA. Calcium chloride is thought to effect some structural alterations in the bacterial cell wall. An efficient method for transformation in Bacillus species involves polyethylene glycol-induced DNA uptake in bacterial protoplasts and subsequent regeneration of the bacterial cell wall. Actinomycetes can be similarly transformed. Transformation can also be achieved by first entrapping the DNA with liposomes followed by their fusion with the host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa.

Isolation of passenger DNA

Passenger DNA may be isolated in a number of ways; the most common of these involves DNA restriction. Restriction endonucleases make possible the cleavage of high-molecular-weight DNA. Although three different classes of these enzymes have been described, only type II restriction endonucleases have been used extensively in the manipulation of DNA. Type II restriction endonucleases are DNAases that recognize specific short nucleotide sequences (usually 4 to 6 base pairs in length), and then cleave both strands of the DNA duplex, generating discrete DNA fragments of defined length and sequence. A number of restriction enzymes make staggered cuts in the two DNA strands, generating single-stranded termini. See also Restriction enzyme.

The various fragments generated when a specific DNA is cut by a restriction enzyme can be easily resolved as bands of distinct molecular weights by agarose gel electrophoresis. Specific sequences of these bands can be identified by a technique known as Southern blotting. In this technique, DNA restriction fragments resolved on a gel are denatured and blotted onto a nitrocellulose filter. The filter is incubated together with a radioactively labeled DNA or RNA probe specific for the gene under study. The labeled probe hybridizes to its complement in the restricted DNA, and the regions of hybridization are detected autoradiographically. Fragments of interest can then be eluted out of these gels and used for cloning. Purification of particular DNA segments prior to cloning reduces the number of recombinant that must later be screened. See also Electrophoresis.

Another method that has been used to generate small DNA fragments is mechanical shearing. Intense personification of high-molecular-weight DNA with ultrasound, or high-speed stirring in a blender, can both be used to produce DNA fragments of a certain size range. Shearing results in random breakage of DNA, producing termini consisting of short, single-stranded regions. Other sources include DNA complementary to poly(A) RNA, or cDNA, which is synthesized in the test tube, and short oligonucleotides that are synthesized chemically.