Identifying Specific Library Sequences

Two types of libraries exist in an organism, depending on the source of the DNA used to make the library. Cloning total DNA from the cell of an organism makes a genomic library. Genomic libraries thus contain all types of sequences, including those which never find their way into messenger RNA (for example, the promoters of genes, or especially, the introns that are found in some or all genes of an organism). On the other hand, cDNA libraries (c stands for copy) are made by first converting mRNA into a DNA copy, a process known as reverse transcription. Then the copy DNA (cDNA) is cloned into a plasmid or bacteriophage vector. Clearly, the probability of isolating a desired DNA sequence in either a cDNA or genomic library depends on the complexity of the DNA source and the number of independent clones.

Specific clones are screened for or selected from the recombinant library. It is relatively simple to see how selection can be done if the desired DNA clone contains a gene required for growth of the host. For example, suppose one wanted to work with the gene sequence that specified an enzyme required for the biosynthesis of leucine. Bacteria that lacked that enzyme would not grow unless the media in which they were growing supplied leucine to the bacteria. If a plasmid library were transformed into those mutant bacteria and the transformants were plated on agar plates that lacked leucine, only the bacteria that contained the cloned DNA sequence encoding the missing enzyme could grow. This is an easy experiment to do but it doesn't always work. In general, the more distantly related the source of the DNA, the more likely that the cloned DNA can be expressed to make a functional product. For example, many other bacteria would contain the enzyme, and the cloned DNA would likely be expressed. On the other hand, DNA from a plant or animal source that encoded the enzyme would likely contain introns and not be expressed in the bacterium.

Hybridization screening uses a nucleic acid probe in an experimental setup very much like a Southern blot. Recombinant bacteria or bacteriophage are grown on a petri plate and then partially transferred to filter paper. The filter paper is then treated to fix the DNA in place, and a specific DNA fragment (or probe) is hybridized to the DNA on the filter paper. The radioactive or otherwise labeled DNA probe sticks to the filter paper only where it contains complementary sequences. Because the blot is like a contact print of the position of the colonies on the petri plate, the location of the complementary sequences serves as the key for amplifying the desired clones. Probes are often made from knowing a small region of the amino acid sequence of a purified protein, figuring out the possible sequences that can encode that amino acid sequence from the genetic code, and then chemically synthesizing all the DNAs that can encode that amino acid sequence. After a single clone is identified, its DNA can be used as a probe to find overlapping clones and the whole assemblage can be fitted into a map of the gene of interest, as shown in Figure 1 .







                  
     Figure 1