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| Modern Genetic Analysis Chapter 10. Recombinant DNA Technology Summary Genetics focuses on the nature of genes, and a major goal is to characterize their structure and function. Recombinant DNA technology has allowed individual genes to be isolated in a test tube and then characterized at the molecular level. The technology is based on restriction enzymes, which cut DNA into defined fragments. Restriction target sites can be mapped and act as DNA landmarks. Restriction fragments often have sticky ends, enabling them to be inserted into a vector capable of replicating in a bacterial cell. Such molecular hybrids are known as recombinant DNA. Bacteria amplify a single recombinant DNA molecule to form a DNA clone. Common vectors are plasmids, phages, and cosmids. An entire genome can be cloned in a set of clones known as a library. A specific clone can be found in a library by using a probe that specifically binds to the DNA or to the protein of the desired clone. Specific clones can also be isolated by their ability to transform null mutants. Tagging is also useful for cloning a gene: transforming DNA or a transposon is used to cause a mutation by insertion, and the DNA adjacent to the tag is isolated. Chromosome walking provides a way of isolating a gene by sequential isolation of overlapping clones, starting from a marker linked to the desired gene. Cloned DNA can be sequenced by several methods, including the arrest of DNA chain growth by dideoxy nucleotides. The polymerase chain reaction uses primers to amplify DNA sequences. It is a way of rapidly isolating DNA whose structure is already partly sequenced and of detecting small amounts of one specific type of DNA. Gel electrophoresis separates different-sized DNA or RNA molecules from a mixture. Probes can detect specific DNA or RNA molecules on the gel, in procedures known as Southern and Northern analyses, respectively.

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