In conventional sense, a library is an information repository that is catalogued for easy information retrieval and use. Biotechnology is also facilitated by a similar system for storage and easy retrieval of genetic information, which can be stored in many formats.
The genes can be decoded into DNA language or can be stored even in their physical form. A genetic library constitutes of cloned DNA segments that belong to an individual. Also, genetic libraries are created according to certain themes including the entire genome, active genes, or a single chromosome.
Cloned fragments of an organism constitute a genomic library. Genomic libraries of various eukaryotes and prokaryotes have been accomplished from various genomic projects.
Genomes are usually large in size, for example, a human genome measures about 3.0 x 10 6 kb. In order to create a genomic library, DNA of an organism is isolated and subjected to restriction enzyme digest. Each DNA fragment is inserted into a specific vector, and each cloned fragment is identified, extracted, sub-cultured, and characterized. Though this is a tedious and painstaking process, there are many modern-day tools and protocols to facilitate the process.
Complementary DNA, also called cDNA, is DNA that is synthesized from its messenger RNA (mRNA) by the process of reverse transcription (against the central dogma). A cDNA molecule is synthesized to represent active genes in a cell. A cDNA library for flowering genes can be constructed during the flowering time. The appropriate mRNA molecules are extracted and poly-A tailed at 3' end. A primer, Poly dT, is added to initiate the DNA synthesis using reverse transcriptase. This process creates a DNA-RNA double-stranded duplex molecule. In order to obtain only the DNA, the RNA strand is removed by using ribonuclease H enzyme or by alkali treatment. The resulting DNA (single-strand) forms the template base for synthesizing a complementary strand. The reaction is catalyzed by the enzyme DNA polymerase I.
A DNA duplex is produced with a close at the 3' end. The single-strand DNA loops back in order to serve as a base to synthesize the complementary strand. The loop can be broken by using S1 nuclease enzyme; the new double-stranded is cloned.
Gene libraries contain thousands to millions of clones. GAATTC acts as the recognition sequence for the EcoRI (restriction endonuclease), heterogeneous bases; the possible arrangement of the bases to form GAATTC is 4,096(4 6). cDNA libraries always remain incomplete because they represent only the reverse transcribed genes. Further, cDNAs are smaller in size when compared to original genes because of the absence of introns.
DNA Colony Hybridization
The method of DNA colony hybridization on a probe and its length varies between 100 to 1000 bp. Probe occurs either natural or synthetic. Natural probes are derived from related organisms and are called heterologous probes. The natural difference that exists between the probes and the target DNA results in a lack of a perfect match during hybridization. A synthetic probe is based on based on amino acid sequence that is obtained from the target gene coded protein.
Genetic libraries are most often created from partial digestions. More than one colony shows a positive signal in autoradiography. In order to determine the clone that contains the target gene, further analysis is required. Techniques such as DNA sequencing, electrophoresis, and restriction endonuclease mapping are some of the best tools employed to identify the complete gene.
Immunological assay screening is similar to a probe screening. Here, an antibody is used instead of a probe. The process is similar to probe hybridization except that proteins are important to immunological assay. After cell lysis, the proteins and the matrix are treated. Since, the DNA consists of 4 with the primary antibody. Following incubation, any unbound antibody is washed away and the secondary antibody specific for the primary antibodies is applied. An enzyme is attached to the secondary antibody (colorless substrate). The enzyme hydrolyses the substrate producing a color compound at the reaction site, thereby identifying the clone harboring the target gene.
One of the major biotechnological activities in use today is the computer-based storage and retrieval of genetic information. This technology gave birth to the scientific discipline called bioinformatics. This knowledge-based discipline aims to predict biological functions using DNA data sequence analysis.
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