5.1 The Exploration of Genes Relies on Key Tools

The recombinant DNA revolution in biology is rooted in the repertoire of enzymes that act on nucleic acids. Restriction enzymes are a key group among them. These endonucleases recognize specific base sequences in double-helical DNA and cleave both strands of the duplex, forming specific fragments of DNA. These restriction fragments can be separated and displayed by gel electrophoresis. The pattern of these fragments on the gel is a fingerprint of a DNA molecule. A DNA fragment containing a particular sequence can be identified by hybridizing it with a labeled single-stranded DNA probe (Southern blotting).

Rapid sequencing techniques have been developed to further the analysis of DNA molecules. DNA can be sequenced by controlled interruption of replication. The fragments produced are separated by gel electrophoresis and visualized by autoradiography of a ³²P label at the 5′ end or by fluorescent tags.

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DNA probes for hybridization reactions, as well as new genes, can be synthesized by the automated solid-phase method. DNA chains as long as 100 nucleotides can be readily synthesized. The polymerase chain reaction makes it possible to greatly amplify specific segments of DNA in vitro. The region amplified is determined by the placement of a pair of primers that are added to the target DNA along with a thermostable DNA polymerase and deoxyribonucleoside triphosphates. The exquisite sensitivity of PCR makes it a choice technique in detecting pathogens and cancer markers, in genotyping, and in amplifying DNA from fossils that are many thousands of years old.

5.2 Recombinant DNA Technology Has Revolutionized All Aspects of Biology

New genes can be constructed in the laboratory, introduced into host cells, and expressed. Novel DNA molecules are made by joining fragments that have complementary cohesive ends produced by the action of a restriction enzyme. DNA ligase seals breaks in DNA chains. Vectors for propagating the DNA include plasmids, λ phage, and bacterial and yeast artificial chromosomes. Specific genes can be cloned from a genomic library with the use of a DNA or RNA probe. Foreign DNA can be expressed after insertion into prokaryotic and eukaryotic cells by the appropriate vector. Specific mutations can be generated in vitro to engineer novel proteins. A mutant protein with a single amino acid substitution can be produced by priming DNA replication with an oligonucleotide encoding the new amino acid. Plasmids can be engineered to permit the facile insertion of a DNA cassette containing any desired mutation. The techniques of protein and nucleic acid chemistry are highly synergistic. Investigators now move back and forth between gene and protein with great facility.

5.3 Complete Genomes Have Been Sequenced and Analyzed

The sequences of many important genomes are known in their entirety. More than 10,000 bacterial and archaeal genomes have been sequenced, including those from key model organisms and important pathogens. The sequence of the human genome has now been completed with nearly full coverage and high precision. Only from 20,000 to 25,000 protein-encoding genes appear to be present in the human genome, a substantially smaller number than earlier estimates. Comparative genomics has become a powerful tool for analyzing individual genomes and for exploring evolution. Genomewide gene-expression patterns can be examined through the use of DNA microarrays.

5.4 Eukaryotic Genes Can Be Quantitated and Manipulated with Considerable Precision

Changes in gene expression can be readily determined by such techniques as quantitative PCR and hybridization to microarrays. The production of transgenic mice carrying mutations known to cause ALS in humans has been a source of considerable insight into the disease mechanism and its possible treatment. The functions of particular genes can be investigated by disruption. One method of disrupting the expression of a particular gene is through RNA interference, which depends on the introduction of specific double-stranded RNA molecules into eukaryotic cells. New DNA can be brought into plant cells by the soil bacterium Agrobacterium tumefaciens, which harbors Ti plasmids. DNA can also be introduced into plant cells by applying intense electric fields, which render them transiently permeable to very large molecules, or by bombarding them with DNA-coated microparticles. Gene therapy holds great promise for clinical medicine, but many challenges remain.

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