Introduction:-
- A DNA library is a collection of clones of DNA designed so that there is a high probability of finding specific piece of the source DNA in the collection.
- There are two types of DNA libraries:-
I. GENOMIC LIBRARY: The genomic library contains DNA fragments representing entire genome of an organism.
II. cDNA LIBRARY: The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell.
I. Genomic library
- A genomic library is a collection of bacterial clones or lysates that when taken together represent every segment of the Genome of an organism.
- The genomic Library Include sequences that are regulatory, specifying for proteins Specifying for structural RNAs and sequences without known function (Junk DNA).
- In the eukaryotic genomic library, the gene sequences contain both the expressed regions (Exons) and non-expressed regions (introns.)
The construction of a genomic Library involves the following steps:-
- Isolation of genomic DNA from suitable tissue.
- Fragmentation of the genomic DNA into fragments of convenient length. This can be carried out either by using the mechanical shearing or by using partial restriction digestion; the mechanical shearing generates the randomly sized fragments that exhibit great variation in the length whereas the partial dilation produces fragments of uniform size.
- Electro phoretic separation of the fragments, the fragments are separated by agarose gel electrophoresis. The separation is based on the size of the fragments, smaller sized fragments migrate faster and to greater distances compare to larger sized fragments. The electro phoretic mobility of a fragment is inversely proportional to the logarithm of its molecular mass
- Cloning of the selected fragments into suitable vectors The initial genomic libraries were constructed by using Lambda replacement vectors such as EMBL3 and EMBL4. The genomic libraries of higher eukaryotes are generated by using high capacity vectors such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) for the lower eukaryotes cosmids are preferred.
- The recombinant vectors containing the genomic DNA fragments are introduced into the cloning host (E. coli K-12 strain that is mutant for restriction modification and recombination)
- After the transfer, the host cell will be screen for the presence of genomic DNA inserts, and the positive clones are collected and are arranged by using chromosome walking method.
- Chromosome walking involves the arrangement of genomic library colonies in the specific order based on the overlapping of the fragments. The chromosome walking technique involves the identification of the homologous fragments at 5' and 3' ends by using hybridization technique with the 5' and 3' probes.
- After the complete analysis of the colony with hybridization based on the homology the colonies are arranged and stored at low temperature
- To determine the complete genome sequence of a given organism.
- To improve the copy number of the genome
- Useful for chromosomal mapping (order of genes on a chromosome)
- It is the source for generation of transgenic animals through genetic engineering.
- Study of the functions of regulatory sequences in vitro
- Study of genetic mutations in various genetic diseases.
- Genomic library is a most useful method in Human Genome Project viii. Germplasm creation for species conservation.
- A collection of bacterial colonies or lysates when taken together represent every cDNA sequence obtained from the reverse transcription of mRNAs from specific tissue. A cDNA library represents only the expressive portion of the genome.
- The cDNAs represent only the coding regions (exons) of the genes since the mRNAs will have only exons. The cDNA libraries are useful in isolating targeting genes that must be expressed in prokaryotic hosts.
- Selection of tissue that has abundance of specific mRNAs.
- Isolation of total cellular RNA: As the from eukaryotic mRNAs are polyadenylated at their 3' ends it is advantage for isolation by affinity chromatography on a poly U Sepharose column or an oligo dT cellulose column.
- Checking of the RNA integrity: Analysis the mRNAs by gel electrophoresis and make sure that the mRNA is not degraded.
- Reverse transcription and generation of double stranded cDNA molecules
- Principles of Gene Manipulation: sixth edition, Sandy B Primrose, Richard M Twyman and Robert W Old.
- https://www.sciencedirect.com/topics/agricultural-and-biologicalsciences/cdnalibraries#:~:text=A%20cDNA%20library%20is%20a,stands%20for%20' complementary').
- https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Bo ok%3A_Basic_Cell_and_Molecular_Biology_(Bergtrom)/15%3A_DNA_T echnologies/15.02%3A_Make_and_Screen_a_cDNA_Library
- https://www.differencebetween.com/difference-between-dna-and-vscdna/
great information about cDNA & Genomic DNA Library
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