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DNA Libraries

DNA library is the collection of DNA fragments of entire or partial genome of an organism that are cloned separately while attached to a suitable vector inside a host cell. DNA library is analogous to an academic library where each clone carrying a particular DNA fragment is similar to a book and the number of cells of a single clonal culture in a culture vessel is analogous to the number of books of that particular book. DNA libraries of a variety of organisms have been constructed, e.g., E. coli, Saccharomyces cerevisiae, Bacillus subtilis, Drosophila (fruit fly), Arabidopsis (a higher plant), etc., which are used for various purposes, like, isolation of a desired gene or DNA fragment, study of the structure of genes, regulation of genes, genome mapping, etc. with the help of specific probes. The DNA library is of the two types:

1.      Genomic DNA Library

Genomic DNA library (gene bank) of an organism is the collection of cloned random DNA fragments covering the entire genome that may contain both introns (do not contain any information of amino acids and after transcription, though, they are present in Hn RNA, but, during processing they are removed to produce mRNA) and exons (sequences containing information required for protein synthesis) in eukaryotes. The genomic DNA library of an organism by shotgun method can be constructed by following the steps:

(i)  Genomic DNA is extracted from the cells of an organism.

(ii) The extracted DNA is cut into random fragments of reasonable size by a particular restriction endonuclease (RE).

(iii) A suitable vector (e.g. a plasmid, a cosmid, etc.) is made linear by cutting with the same restriction enzyme to get the same complementary ends as in the genomic DNA fragments.

(iv) The two types of linear DNAs (vector and individual genomic DNA fragments) are mixed together in a culture vessel and the complementary ends of the recombinant
DNA, thus, obtained are joined together by adding the DNA ligase enzyme.

(v) These recombinant DNAs are separately introduced into a host cell (e.g. Escherichia coli) and cloned by growing in artificial nutrient medium in a culture vessel.

(vi) Thus, the individual transformed cells, each containing a genomic DNA fragment, are
inoculated at the surface of solidified nutrient medium in a Petri plate and allowed to grow into separate colonies (clones). Individual colonies can also be transferred into the liquid medium in separate culture vessels. The set of cloned DNA fragments from the entire genome of an organism maintained separately in host cells constitutes the genomic DNA library. The number of cloned DNA fragments in a genomic DNA library increases with increase in the size of genome and the human genome comprises more than 8,00,000 cloned fragments (Fig, 12.18),

2.    cDNA Library

Many organisms have very large genome and the construction of genomic DNA library in such organisms would require maintenance and screening of a large number of clones. Moreover, the fragments cloned in genomic DNA library may include introns as well that should be removed before the translation process of protein synthesis as they do not contain any information for amino acids. When such bacterial clones are utilized for the commercial production of a particular protein, the transformed host bacteria do not possess the machinery required for the processing of the HnRNA heterogeneous RNA) transcribed from that particular split gene to produce mRNA. These problems can be overcome by constructing the cDNA libraries.

 cDNA is the complementary DNA (copy DNA) of mRNA that lacks introns and other undesirable nucleotide sequences. Thus, cDNA library of an organism contains only the expressed genes (cDNAs) as they are prepared from the mRNA by the following steps:

(i)  Extraction of mRNAs from the cells (in unicellular organisms) or different tissues
engaged in active synthesis of proteins (in multicellular higher organisms, different genes are expressed in various tissues to give different types of mRNAs and proteins due to regulation of gene expression) to get all the possible kinds of mRNAs of an organism.

Fig-12.18

Fig-12.18

Fig 12.18 Technique of genomic DNA library formation (RE1=Restriction enzyme 1)

 (ii)Addition of a primer that is made up of a short sequence of dT, causes base pairing with the complimentary sequence of poly. A tail found at the 3’ end of mRNA

(iii)Addition of reverse transcriptase enzyme produces cDNA strand (complimentary to mRNA) by copying mRNA as the template and adding dNTPs at the 3’ end of the primer. Thus, a double-stranded mRNA-cDNA complex is found.

(iv)Treatment with alkali or the ribonuclease enzyme (that breaks down RNAs) helps in the removal of mRNA from this double-stranded complex to form single-stranded cDNA.

(v)Now, the added DNA polymerase utilizes cDNA as template to give double strand cDNA molecule.

(vi)The hook at one end of this cDNA is removed by S1 nuclease.

(vii)A suitable vector DNA is cut with a restriction enzyme producing blunt ends (not complementary sticky ends).

(viii)Now, the cDNA and linearised vector DNA are mixed separately with a linker DNA (short double-stranded DNA molecules which are actually the restriction sites of restriction enzyme).

(ix)Addition of T4 DNA ligase (obtained from T4 bacteriophage which can ligate two double stranded DNA molecules having blunt ends) leads to the ligation of linker DNAs at the ends of cDNA and linear vector DNAs.

(x)The two DNAs are treated separately with the restriction enzyme that recognizes and cuts the attached linker DNAs to produce complementary sticky ends.

(xi)Mixing of the two DNAs leads to the construction of recombinant DNA that is introduced into a suitable host cell. Thus, the set of all the host cells transformed with a particular cDNA, constitutes a cDNA library (Fig. 12.19).

Fig-12.19

Fig-12.19

Fig-12.19

Fig. 12.19 Technique for the preparation of cDNA library.

Colony Hybridization Technique

The genomic/cDNA library can be perpetuated indefinitely and, whenever required, the desired DNA fragment, after identification by the colony hybridization method, can be retrieved using the same restriction enzyme that was used for making the recombinant DNA. In colony hybridization technique for screening of the desired clone from the genomic!cDNA library, following steps can be implemented:

        i.            From the master plate, containing the colonies of transformed bacterial cells constituting the genomic/cDNA library, replica plate is made which is the impression of master plate with respect to the positions of colonies of various clones.

      ii.            The DNAs of individual colonies are denatured (made single-stranded) in situ when DNA still inside the cells.

    iii.            The denatured DNAs are then southern blotted on a nitrocellulose membrane.

iv.            The single-stranded DNA is then hybridized with a specific radiolabelled probe (small DNA or RNA molecules, generally single stranded) to identify the clone.

v.            Then it is autoradiographed by covering and exposing the membrane with an X -ray film in dark.

vi.            The identified colonies are then retrieved from the corresponding positions on the master plate (Fig. 12.20).

Fig-12.20

Fig-12.20

Fig. 12.20 Colony hybridization method for the selection of desire from genomic/cDNA library.