Recombinant restriction-enzyme analysis

Because the use of PCR very often results in the introduction of unwanted mutations, it was decided to construct die whole P putida pepK gene from these two clones without PCR. First, the insert fragments of both recombinant clones were isolated and fused in vector pGEM5Zf(+) (Promega). A plasmid with the two insert fragments in correct order and orientation was then identified by restriction enzyme analysis. This plasmid contains the complete P. putida pepA gene without any mutation. 

Workers in the early 1970s recognized that restriction enzymes provided tools not only for DNA mapping but also for constmction of new DNA species not found in nature. A collection of recombinant DNA species consisting of many passenger sequences joined to identical vector molecules is called a hbrary. Individual recombinant DNAs are isolated from single clones of the Hbrary for detailed analysis and manipulation. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

Experiment 14 introduced students to some principles and techniques involved in recombinant DNA research. Specifically, the experiment outlined the replication, isolation, and analysis of bacterial plasmid vehicles for molecular cloning experiments. This experiment describes another tool that is essential in hybrid plasmid construction and analysis-restriction enzyme action. The procedures introduced here have also found widespread use in the analysis and characterization of all DNA molecules. 

The multitude of PIBs produced during this process are composed of both types of input nucleocapsids. These progeny PIBs can be fed to caterpillars, initiating an infection of both virus types. The nature of the growth dynamics between the virus types can be followed in two ways. The PIBs themselves can be dissolved and their nucleocapsid components analyzed by DNA restriction enzyme digestion and subsequent filter hybridization analysis. This allows an assessment of the proportions of wild type to recombinant virus in the MC-PIB. Also, the free NOVs present in an infected caterpillar can be collected and analyzed by plaquing in cell culture. This provides an assessment of the frequency of wild type to recombinant virus free in the host. 

The use of restriction enzymes to cleave DNA at specific sequences was mentioned earlier in this chapter in the context of DNA sequence analysis. These enzymes are also important in the field of recombinant DNA technology. We will illustrate this application by describing a method for the production of human insulin. 

The combined genomics and chemical approaches to plant terpenoid research are not restricted to the few plant species for which more or less complete genome sequences are now available. The discovery of many of the genes and enzymes for the formation of terpenoids such as menthol and related monoter-penes in peppermint Mentha x piperita) (15), artemisinin in Artemisia annua (16), Taxol in the yew tree (Taxus) (17), or conifer diterpene resin acids in species of spmce (Picea ) and pine (Pinus) (18) have been possible on the foundation of highly specialized efforts of EST and full-length cDNA sequencing combined with characterization of recombinant enzymes and analysis of the terpenoid metabolome of the target plant species. 

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