Genomic library

Collection of the total genomic DNA from a single organism
A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism commonly a population of Escherichia coli or yeast with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis. There are several kinds of vectors available with various insert capacities. Generally, libraries made from organisms with larger genomes require vectors featuring larger inserts, thereby fewer vector molecules are needed to make the library. Researchers can choose a vector also considering the ideal insert size to find the desired number of clones necessary for full genome coverage. Genomic libraries are commonly used for sequencing applications. They have played an important role in the whole genome sequencing of several organisms, including the human genome and several model organisms.

The first DNA based genome ever fully sequenced was achieved by two time Nobel Prize winner, Frederick Sanger, in 1977. Sanger and his team of scientists created a library of the bacteriophage, phi X 174, for use in DNA sequencing. The importance of this success contributed to the ever increasing demand for sequencing genomes to research gene therapy. Teams are now able to catalog polymorphisms in genomes and investigate those candidate genes contributing to maladies such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, rheumatoid arthritis, and Type 1 diabetes. These are due to the advance of genome wide association studies from the ability to create and sequence genomic libraries. Prior, linkage and candidate gene studies were some of the only approaches.

Construction of a genomic library involves creating many recombinant DNA molecules. An organism's genomic DNA is extracted and then digested with a restriction enzyme. For organisms with very small genomes (~10 kb), the digested fragments can be separated by gel electrophoresis. The separated fragments can then be excised and cloned into the vector separately. However, when a large genome is digested with a restriction enzyme, there are far too many fragments to excise individually. The entire set of fragments must be cloned together with the vector, and separation of clones can occur after. In either case, the fragments are ligated into a vector that has been digested with the same restriction enzyme. The vector containing the inserted fragments of genomic DNA can then be introduced into a host organism. Below is a diagram of the above outlined steps.

After a genomic library is constructed with a viral vector, such as lambda phage, the titer of the library can be determined. Calculating the titer allows researchers to approximate how many infectious viral particles were successfully created in the library. To do this, dilutions of the library are used to transform cultures of E. coli of known concentrations. The cultures are then plated on agar plates and incubated overnight. The number of viral plaques are counted and can be used to calculate the total number of infectious viral particles in the library. Most viral vectors also carry a marker that allows clones containing an insert to be distinguished from those that do not have an insert. This allows researchers to also determine the percentage of infectious viral particles actually carrying a fragment of the library. A similar method can be used to titer genomic libraries made with non viral vectors, such as plasmids and BACs. A test ligation of the library can be used to transform E. coli. The transformation is then spread on agar plates and incubated overnight. The titer of the transformation is determined by counting the number of colonies present on the plates. These vectors generally have a selectable marker allowing the differentiation of clones containing an insert from those that do not. By doing this test, researchers can also determine the efficiency of the ligation and make adjustments as needed to ensure they get the desired number of clones for the library.

In order to isolate clones that contain regions of interest from a library, the library must first be screened. One method of screening is hybridization. Each transformed host cell of a library will contain only one vector with one insert of DNA. The whole library can be plated onto a filter over media. The filter and colonies are prepared for hybridization and then labeled with a probe. The target DNA insert of interest can be identified by detection such as autoradiography because of the hybridization with the probe as seen below. Another method of screening is with polymerase chain reaction (PCR). Some libraries are stored as pools of clones and screening by PCR is an efficient way to identify pools containing specific clones.

Genome wide association studies are general applications to find specific gene targets and polymorphisms within the human race. In fact, the International HapMap project was created through a partnership of scientists and agencies from several countries to catalog and utilize this data. The goal of this project is to compare genetic sequences of different individuals to elucidate similarities and differences within chromosomal regions. Overall, advances in genome library construction and DNA sequencing has allowed for efficient discovery of different molecular targets. Assimilation of these features through such efficient methods can hasten the employment of novel drug candidates.