Use of synthetic oligonucleotides in gene isolation and manipulation.
Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008099:Liver / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D013917:Thrombin / Q000037:antagonists & inhibitors; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000494:pharmacology
D003062:Codon; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D000515:alpha 1-Antitrypsin; D004247:DNA; D010196:Pancreatic Elastase; D013917:Thrombin