A gene library is the collection of different DNA sequence 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 bases of source of DNA used: (1) genomic library (where genomic DNA is used), and (2) cDNA library (where cDNA or complimentary DNA is produced using mRNA). A gene library should contains a certain number of recombinants with a high probability of containing any particular sequence. This value can be calculated when you know the genome size and average size of inserted into the vector.
The probability of getting a given gene sequence in a gene library, the number (N) of recombinants ( bacterial colonies or viral plaques) can be calculated by using following formula:
Where, P = the desired probability and f = the fraction of one genome in one
insert. For a probability of 0.99 with insert size 2× 104 bp, the value of genome for
E.coli (4.6 × 106bp) will be:
NE.Coli = 〖(In (1-0.99))/(In ⌊(1-(2×(20^4)/4.6×10^6)⌋ )〗^ = 1.1 ×10^3
1. 1. Genomic library
It is a collection of clones that
represent the complete genome of an organism. All fragments of DNA are inserted
into vector for further propagation into suitable host represent the entire
genome of an organism. For construction of genomic library the entire genomicDNA is isolated from host cell/tissue, purified and broken randomly into fragments
of correct size for cloning into a suitable vector. We have two basic ways of
fragmenting genomic DNA randomly: physical
shearing (e.g. pipetting, mixing or sonication)
and partial restriction enzyme digestion
(by using limited amount of restriction enzyme the DNA is not digestion at
every recognition sequence). Using these method genomic DNA is broken randomly
into smaller fragments.
Fig. shows a total of 6 random DNA fragment obtained after physical sharing. The
vector isolated from bacterium is also digested with the restriction enzyme
which digest genomic DNA. The fragment of genomic DNA is inserted into the
vector. Each vector consists of different fragments of DNA. The recombinant DNA
molecule are transferred into the bacterial cell or bacterial cell or
bacteriophage particles are assembled.
The genomic library for organisms with
smaller genomic size (e.g. E.Coli) can be constructed in plasmid vector. Only 5,000
clones (of the average size of 5,000 bp) result in 99% chance of cloning the
entire genome of 4.6 × 106 bp. In addition, library from
organisms with larger genomes are constructed using phage l, cosmid, BAC or YAC
vector.
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