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A DNA Library

Within the past few years, the technologies of recombinant DNA have mushroomed. We will follow a typical sequence of procedures that might be used to solve a particular problem or to produce a specific product.

The first task in recombinant DNA technology is to produce a DNA library – a readily accessible, easily duplicable assemblage of all the DNA of a particular organism. The entire set of genes carried by a member of any given species is called a genome. Why build a DNA library of a species’ genome? A DNA library organizes the DNA in a way that researchers can use it. restriction enzymes, plasmids, and bacteria are the most commonly used tools in assembling a DNA library.

Many bacteria produce restriction enzymes, which sever DNA at particular nucleotide sequences. In nature, restriction enzymes defend bacteria against viral infections by cutting apart the viral DNA. (The bacteria protect their own DNA, probably by attaching methyl groups to some of the DNA nucleotides.) Researchers have isolated restriction enzymes and use them to break DNA into shorter strands at specific sites.

Most restriction enzymes recognize and sever palindromic sections of DNA, in which the nucleotide order is the same in one direction on one strand as in the reverse direction on other strand. (A palindrome is a word that reads the same forward and backward, such as “madam”.) These single-stranded cut pieces of the DNA fragment are called ‘sticky ends’, because they will stick to (form hydrogen bonds with) other single-stranded cut pieces of DNA with the complementary series of bases. If the appropriate DNA repair enzyme (called DNA ligase) is added, DNA from different sources cut by the same restriction enzyme can be joined as if the DNA had occurred naturally. Segments of DNA from fundamentally different types of organisms, such as bacteria and humans, can be joined if they have complementary sticky ends.

Many different restriction enzymes have been isolated from various species of bacteria. Each cuts DNA apart at different but specific palindromic nucleotide sequences. The variety of restriction enzymes has enabled molecular geneticists to identify and isolate specific segments of DNA from many organisms, including humans.

Suppose now that human DNA is isolated from white blood cells and is cut apart into many small fragments with a restriction enzyme. The same restriction enzyme is then used to sever the DNA of bacterial plasmids. Now both human and plasmid DNA have complementary sticky ends that, when mixed, form hydrogen bonds. When DNA ligase is added, it bonds the sugar-phosphate backbones together, inserting segments of human DNA into plasmids.

The new rings of plasmid-human DNA (recombinant DNA) are mixed with bacteria, which take up the recombinant DNA. Millions or billions of plasmids collectively could incorporate DNA from the entire human genome. Usually, 100 to 1000 times more bacteria than plasmids are used, so that no individual bacterium ends up with more than one recombinant DNA molecule. The resulting population of bacteria containing recombinant plasmid-human DNA constitutes a human DNA library.

Unit 4

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