Recombinant DNA is constructed in the laboratory to allow researchers to amplify and analyze DNA segments (donor DNA) from any genome or from DNA copies of mRNAs. Three sources of donor DNA are (1) the entire genome digested with a restriction enzyme, (2) PCR products of specific DNA regions defined by the flanking primer sequences, and (3) cDNA copies of mRNAs.

The polymerase chain reaction is a powerful method for the direct amplification of a relatively small sequence of DNA from within a complex mixture of DNA, without the need for a host cell or very much starting material. The key is to have primers that are complementary to flanking regions on each of the two DNA strands. These regions act as sites for polymerization. Multiple rounds of denaturation, priming, and polymerization amplify the sequence of interest exponentially.

To insert donor DNA into vectors, donor DNA and vector DNA are cut by the same restriction endonuclease at specific sequences. Vector and donor DNA are joined by annealing the sticky ends that result from digestion, followed by ligation to covalently join the molecules. PCR and cDNA molecules are inserted into vectors by first adding restriction-endonuclease-recognition sequences to the 5′ end of PCR primers or by ligating short adapters containing restriction sites to their ends before insertion into the vector.

There are a wide variety of bacterial vectors. The choice of vector depends largely on the size of the DNA fragment to be cloned. Plasmids are used to clone small restriction fragments, PCR molecules, or cDNA molecules. Intermediate-size fragments, such as those resulting from the digestion of genomic DNA, can be cloned into modified versions of λ bacteriophage (for inserts of 10–15 kb) or into phage-plasmid hybrids called fosmids (for inserts of 35–45 kb). Finally, bacterial artificial chromosomes (BACs) are used routinely to clone very large genomic fragments (~100–200 kb).

The vector-donor DNA construct is amplified inside bacterial host cells as extrachromosomal molecules that are replicated when the host is replicating its genome. The result of amplification of plasmids, phages, and BACs is clones containing multiple copies of each recombinant DNA construct. In contrast, only a single fosmid is present in each bacterial cell.

Often, finding a specific clone with a gene of interest requires screening a full genomic library. A genomic library is a set of clones, ligated in the same vector, that together represent all regions of the genome of the organism in question. The number of clones that constitute a genomic library depends on (1) the size of the genome in question and (2) the insert size tolerated by the particular cloning-vector system. Similarly, a cDNA library is a representation of the total mRNA set produced by a given tissue or developmental stage in a given organism.

Labeled single-stranded DNA or RNA probes are important for fishing out similar or identical sequences from complex mixtures of molecules, either in genomic or cDNA libraries or in Southern (DNA) and Northern (RNA) blotting. The general principle of the technique for identifying clones or gel fragments is to create an “image” of the colonies or plaques on an agar petri-dish culture or of the nucleic acids that have been separated in an electric field passed through a gel matrix. The DNA or RNA is then denatured and mixed with a denatured probe that has been labeled with a fluorescent dye or a radioactive label. After the unbound probe has been washed off, the location of the probe is detected either by observing its fluorescence or, if radioactive, by exposing the sample to X-ray film. The locations of the probe correspond to the locations of the relevant DNA or RNA in the original petri dish or electrophoresis gel. Labeled antibodies are important probes for fishing out specific proteins from complex mixtures produced either by expression libraries (with cDNA inserts) or in Western blotting.

Vast genomic resources are making it increasingly possible to isolate genes solely from knowledge of their position on a genetic map. Two overall procedures are forward genetic strategies called positional cloning and fine-structure mapping. With the sequencing of the human genome and the availability of families with inherited disorders, fine-structure-mapping strategies have led to isolation of genes that when mutated produce human disease.

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Transgenes are engineered DNA molecules that are introduced and expressed in eukaryotic cells. They can be used to engineer a novel mutation or to study the regulatory sequences that constitute part of a gene. Transgenes can be introduced as extrachromosomal molecules or they can be integrated into a chromosome, either in random (ectopic) locations or in place of the homologous gene, depending on the system. Typically, the mechanisms used to introduce a transgene depend on an understanding and exploitation of the reproductive biology of the organism.