SUMMARY

• 41.1 Nucleic Acids Can Be Synthesized from Protein-Sequence Data

  Amino-acid-sequence information obtained from the purified receptor can be used to synthesize DNA probes that correspond to the amino acid sequence. The technique is to add modified deoxynucleotides to one another to form a growing strand that is linked to an insoluble support. DNA strands as long as 100 nucleotides can be readily synthesized.

• 41.2 Recombinant DNA Technology Has Revolutionized All Aspects of Biology

  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, displayed by gel electrophoresis, and inserted in vectors to generate recombinant DNA molecules.

• 41.3 Eukaryotic Genes Can Be Manipulated with Considerable Precision

  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 and λ phage. With the use of these techniques, cDNA libraries for any tissue and genomic libraries from any organism can be generated. These libraries can then be screened by using DNA probes. Foreign DNA can be expressed after insertion into bacteria by the appropriate vector. Specific genes can be cloned from a genomic library by using a DNA probe.

  Rapid sequencing techniques have been developed to further the analysis of DNA molecules. DNA can be sequenced by the 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.

  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.

  DNA microarrays, or gene chips, allow the detection of changes in the transcription of hundreds of genes simultaneously. Microarray analysis shows how transcription responds to various physiological and pathological conditions, such as the presence of a hormone or the transformation into a cancerous state.