Gene libraries are used to find new genes, to sequence entire genomes, and to compare genes from different organisms. Gene libraries are made when the entire DNA from one particular organism is digested into fragments using restriction enzymes, and then each of the fragments is cloned into a vector and transformed into an appropriate host.
The basic steps used to construct a library are:
1 . Isolate the chromosomal DNA from an organism, such as E. coli , yeast, or humans.
2 . Digest the DNA with one or two different restriction enzymes.
3 . Linearize a suitable cloning vector with the same restriction enzyme(s).
4 . Mix the cut chromosome fragments with the linearized vector and ligate.
5 . Transform this mixture into E. coli .
6 . Isolate large numbers of E. coli transformants.
The type of restriction enzyme affects the type of library. Because restriction sites are not evenly spaced in the genome, some inserts will be large and others small. Using a restriction enzyme that recognizes only four base pairs will give a mixture of mostly small fragments, whereas a restriction enzyme that has a six or eight base-pair recognition sequence will generate larger fragments. (Note that finding a particular four base-pair sequence in a genome is more likely than finding a six base-pair sequence.)
Even if an enzyme that recognizes a four base-pair recognition sequence is used to digest the entire genome, there may still be segments that are too large to be cloned. Conversely, clustered restriction sites will cause some genes will be cut into several pieces. To avoid this, partial digestion is often used.
The enzyme is allowed to cut the DNA for only a short time, and many of the restriction enzyme sites are not cut, leaving larger pieces for the library. In addition, it is usual to construct another library using a different restriction enzyme.
SCREENING THE LIBRARY OF GENES BY HYBRIDIZATION:
Once the library is assembled, researchers often want to identify a particular gene or segment of DNA within the library. Sometimes the gene of interest is similar to one from another organism. Sometimes the gene of interest contains a particular sequence.
For example, many enzymes use ATP to provide energy. Enzymes that bind ATP share a common signature sequence whether they come from bacteria or humans. This sequence can be used to find other enzymes that bind ATP. Such common sequence motifs may also suggest that a protein will bind various cofactors, other proteins, and DNA, to name a few examples.
Screening DNA libraries by hybridization requires preparing the library DNA and preparing the labeled probe. A gene library is stored as a bacterial culture of E. coli cells, each having a plasmid with a different insert. The culture is grown up, diluted, and plated onto many different agar plates so that the colonies are spaced apart from one another. The colonies are transferred to a nylon filter and the DNA from each colony is released from the cells by lysing them with detergent. The cellular components are rinsed from the filters. The DNA sticks to the nylon membrane and is then denatured to form single strands.
If a scientist is looking for a particular gene in the target organism, the probe for the library may be the corresponding gene from a related organism. The probe is usually just a segment of the gene, because a smaller piece is easier to manipulate. The probe DNA can be synthesized and labeled either with radioactivity or with chemiluminescence. The probe is heated to make it single-stranded and mixed with the library DNA on the nylon filters. The probe hybridizes with matching sequences in the library. The level of match needed for binding can be adjusted by incubating at various temperatures. The higher the temperature, the more stringent, that is, the more closely matched the sequences must be. The lower the temperature, the less stringent. If the probe is labeled with radioactivity, photographic film will turn black where the probe and library DNA hybridized. The black spot is aligned with the original bacterial colony. Usually the most likely colony plus its neighbors are selected, grown, plated, and rescreened with the same probe to ensure that a single transformant is isolated. Then the DNA from this isolate can be analyzed by sequencing