Genomic DNA library

 

                                             Genomic DNA library

A collection of clones containing all DNA segments of genome of an organism is called genomic DNA library.It is created by inserting all fragments of  genomic DNA in to host cells using vectors.

 

(i) Isolation of DNA: The genomic DNA is isolated from the source organism using a standard procedure.

 

(ii) Restriction digestion:  The isolated genomic DNA is cut with a restriction enzyme to generate small DNA fragments. While cutting the DNA with a single restriction enzyme, longer fragments may be generated. Hence, random cuts are made with two different restriction enzymes. This method is called Maniatis strategy.

 

(iii) Gel Electrophoresis: The restriction digest is electrophoresed on a polyacrylamide gel in the presence of  molecular weight markers in one lane.

 

(iv) Selection of clonable DNA fragment: DNA fragments of clonable size are isolated from the gel and the smaller as well as larger fragments are discarded. For example, if the DNA fragments have to be cloned in plasmids, size of the fragments should be between 5 and l5 kb; so fragments lesser than 5 kb and larger than 15 kb are discarded.

 

(v) Removal of 5^/ phosphate group: The isolated DNA fragments are treated with alkaline phosphatase to remove 5^/ -phosphate groups from the ends of  the DNAs.

 

(vi) Construct r-DNA: The vector DNA is cleaved with one or two restriction enzymes which were used to digest the genomic DNA.The dephosphorylated DNA fragments are mixed with the linearized vector DNA to construct r-DNAs.

 

(vi) Transformation in E. coli: The rDNAs thus constructed are introduced into E. coli or yeast cells. If the DNA in plasmids, they are introduced into means of transformation. If the DNA in cosmids, λ-DNA or phagemids,transducing virus particles are developed to deliver the rDNAs into E.coli. DNAs cloned in PAC and BAC are delivered into E. coli by transformation using electroporation. If the DNA is cloned in YAC, transfection with gentle electroporation is used to deliver the DNAs into yeast cells.

 

(vii) Selection of recombinant E. coli:  The recombinants are selected by using appropriate methods fitted for the selected vector.

 

(viii) Screening of recombinant E. coli:The recombinant cells are separated from one another using dilution plating and subsequent culture.The microbial cultures are screened to detect the contigs.Based on the contigs analysis, the clones are arranged in the genome library. Screening of DNA Library .

 

                                     Analysis of various clones to arrange them into contigs is called screening of genomic library. The clones are arranged into contigs based on the principle of overlapping between the adjacent clones. The continuous clones representing the entire genomic DNA of an organism in the correct order are called Contigs. Restriction fragment fingerprinting, chromosome walking, repetitive DNA fingerprinting and STS (Sequence tagged sites) mapping are used to arrange the clones into contigs.

 

 

 

 

 

 

 

 

 

 

 

Importance of genomic library:

 

1. Genomic libraries provide DNA fragments of organisms for sequencing projects.

2. Gene of interest can be obtained from the genomic DNA library for gene manipulation works.

3. Genomic libraries help for the construction of physical maps of organisms.

4. Genomic libraries are useful to study silent genes that do not express in the source organism.

5. Genomic libraries simplify the molecular  research tasks by providing specimens

 researchers all over the world.