A gene library is the collection of different DNA sequences from an organism where each sequence has been cloned into a vector for ease of purification, storage and analysis.
There are two types of libraries on the basis of source of DNA used (i) genomic library (where genomic DNA is used), and (ii) cDNA library (where cDNA or complementary DNA is produced using mRNA).
A gene library should contain a certain number of recombinants with a high probability of containing any particular sequence. This value can be calculated if you know the genome size and average size of insert in the vector.
The probability of getting a given sequence in a gene library, the number (N) of recombinants (bacterial colonies or viral plaques) can be calculated by using the following formula:
1. Genomic Library :
It is a collection of clones that represent the complete genome of an organism. All fragments of DNA inserted into vectors for further propagation into suitable host represent the entire genome of an organism.
For construction of a genomic library the entire genomic DNA is isolated from host cells/ tissues, purified and broken randomly into fragments of correct size for cloning into a suitable vector.
There are two basic ways of fragmenting genomic DNA randomly: physical shearing (e.g. pipetting, mixing or sonication) and partial restriction enzyme digestion (by using limiting amount of restriction enzyme the DNA is not digested at every recognition sequence). Using these methods genomic DNA is broken randomly into smaller fragments.
It shows a total of 6 random DNA fragments obtained after physical shearing. The vector isolated from bacterium is also digested with the same restriction enzyme which digests genomic DNA.
The fragments of genomic DNA are inserted into the vector. Each vector consists of different fragments of DNA. The recombinant DNA molecules are transferred into bacterial cells or bacteriophage particles are assembled.
The genomic library for organisms with smaller genome size (e.g. E. coli) can be constructed in plasmid vector. Only 5,000 clones (of the average size of 5,000 bp) results in 99% chance of cloning the entire genome of 4.6×106 bp. In addition, libraries from organisms with larger genomes are constructed using phage X, cosmid, BAC or YAC vectors.
2. cDNA Library
Reverse transcriptase a single strand of cDNA is synthesised on mRNA template by using a primer oligo (dT) (Fig. 3.12). Alkali hydrolysis by NaOH separates single stranded cDNA from mRNA.
Oligo (dG) is used to prime synthesis of second strand by using either reverse transcriptase or Klenow fragment of E. coli DNA polymerase I. This results in formation of cDNA complex.
Special nucleic acid linkers and restriction enzyme are added to create sticky ends in cDNA duplex for cloning in vector. The recombinant DNA molecules are incorporated into host cells.