Genomic DNA Library Construction - Techniques of Biotechnology and Innovations

GENOMIC DNA LIBRARY CONSTRUCTION

The collections of recombinant DNA molecule carrying the insert of genomic DNA fragment of organism, the sum of total DNA insert of this collection represents the entire genome of the respective organism. For the library construction entire genomic DNA of an organism subjected for physical sharing or enzymes based digestion with the intention to produce random fragments. The partial digestion of genomic DNA is performed with frequent cutter restriction endonuclease, mainly 4 bp cutters. It is assumed that the recognition sequence of restriction enzyme arranged randomly hence chance of occurrence of 4 bp restriction enzymes site is once in 4⁴= 256 bp. The cloning of the small fragment leads to achieve high rate cloning efficiency with such enzymepartial digestion performed either by giving half of the normal enzyme quantity or reducing the time of incubation to half of the normal timing. The produced fragment are cloned into the suitable vectors (plasmid/ phage based vectors) depending upon the size of the fragment produced. In general, lambda based or cosmid vectors are used to clone the insert size of 23-25 Kb and 50 Kb respectively. The recombinant molecules are transferred in to an appropriate host. The transformant number may vary and depend on size of the fragment produced from intact genome. Hence, size of the library directly proportional to number of fragment produced. Further, it depends on type of restriction enzyme used. For example, genome size of an organism is 1,00,000 bp and 4 bp cuter is using for genome library construction. Then, number of clones produced is directly proportional to genome size and inversely to size of fragment produced from restriction enzyme. For 4 bp cutter total 340 cones can be produced from 1, 00,000 bp genomic DNA. Apart, the cloning efficiency and probability of insert representation in the library cost the library size.