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<h2 style="text-align: justify;">1. Genomic Library </h2>
The genomic library represents a collection of the entire genome of an individual animal, bacteria, plant, or virus and is used for the wider study. It may also be called the collection of cloud segments of DNA containing at least one copy of, every gene from a particular organism.
<h3 style="text-align: justify;">Construction of the genomic library </h3>
Step 1 DNA isolation: isolate complete DNA from any cell under study.
Step 2 Cutting isolated DNA with restriction fragments of suitable size: Restriction enzymes like Eco RI are used to cut into fragments into a suitable size.
Step 3 Incorporation of this fragment to a suitable vector-like plasmid, cosmid, etc leading to the formation of rDNA molecule.
Step 4 Introduction of rDNA molecule into the suitable host as E.coli. The plasmid multiplies inside the host forming numerous copies.&nbsp;
<img style="display: block; margin-left: auto; margin-right: auto;" src="https://vediphlie-aws.s3.ap-south-1.amazonaws.com/Construction_of_genomic_library_da82bf8ba5.PNG" alt="Construction of genomic library" />
Fig.: Construction of genomic library.
Step 5 Screening libraries: A common method of screening plasmid-based genomic libraries is to carry out a colony, hybridization experiment.
1) Host bacteria containing plasmid-based or bacteriophage-based libraries are plated out on a Petri dish and allowed to grow overnight to form colonies/plaques.
2) A replica of the bacterial colonies (or plaques) is then obtained by overlaying the plate with a nitrocellulose disc membrane.
3) The membrane is removed, treated with alkali to dissociate bound DNA duplexes into single-strand DNA, dried &amp; placed in a sealed bag with a labeled probe. (recombinant DNA probe).
4) Any DNA sequences complementary to probe DNA will be revealed by autoradiography of the nitrocellulose disc. Bacterial colonies (phage plaques) containing clones bearing target DNA are identified on the film and can be recovered from the master plate.
<img style="display: block; margin-left: auto; margin-right: auto;" src="https://vediphlie-aws.s3.ap-south-1.amazonaws.com/Screening_of_genomic_library_a1b5848b90.PNG" alt="Screening of genomic library" />
Fig.: Screening of genomic library.
<h2 style="text-align: justify;">Advantage of Genomic Library </h2>
Identification of a clone encoding a particular gene of interest. It is useful for prokaryotic organisms having relatively small genomes. Genomic libraries from the eukaryotic organisms are very important to study the genome sequence of a particular gene including its regulatory sequence and its pattern of introns and exons.
No of clone required for a library.
f = genome size / fragment size
for E.coli&nbsp;⟶
genome size = 4.6 Mb
average cloned fragment = 5 Kb
f = 4.6 x 106 / 5 x 103 = 920
Actual no of clones required
N = ln (1&ndash;p) / ln (1 &ndash; 1/f)
Where N = number of clone
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; P = Probability that a given gene will be present. &nbsp;
<h2 style="text-align: justify;">2. cDNA Library -</h2>
The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. cDNA (complementary DNA) is synthetic DNA made from mRNA with the use of enzyme reverse transcriptase Because it is made from mRNA, cDNA is devoid of both upstream and downstream regulatory sequences and of introns. The double-stranded cDNA molecules synthesized by the activity of reverse transcriptase and DNA polymerase can be inserted into a plasmid or virus vector and cloned. Each clone obtained in this way is called a cDNA clone, obtained in this way is called a cDNA clone, and the entire collection of clones derived from an mRNA preparation constitutes a cDNA library.&nbsp;
<img style="display: block; margin-left: auto; margin-right: auto;" src="https://vediphlie-aws.s3.ap-south-1.amazonaws.com/C_DNA_library_32ba752d60.PNG" alt="C-DNA library" />
Fig.: C-DNA library.
<h2 style="text-align: left;">3. Differences between Genomic Library &amp; cDNA Library -</h2>
<img style="display: block; margin-left: auto; margin-right: auto;" src="https://vediphlie-aws.s3.ap-south-1.amazonaws.com/Differences_between_Genomic_Library_and_c_DNA_Library_ae964931ea.PNG" alt="Differences between Genomic Library &amp; cDNA Library" />
<h2>4. Isolation of mRNA -</h2>
In eukaryotes, gene expression is tissue-specific for ex. the gene encoding globin proteins are expressed only in erythrocyte precursor cells called reticulocytes using this information a target gene can be cloned by isolating the mRNA from a specific tissue.
m&ndash;RNA can be isolated by
Affinity chromatography
Spinning down m-RNA by density gradient centrifugation.
The 3' end of m-RNA consists of 50-250 adenylate residue (Poly-Atail) so it can separate easily by oligo dT on resin.
A complementary DNA is synthesized from m&ndash;RNA with the help of RTc.
RTc has three special features of catalyst.
1. RNA dependent DNA polymerase
2. RNase
3. DNA-dependent DNA polymerase.
This RTc obtains from the avian myeloblastosis virus.
RNase&ndash;H removes &ndash; mRNA from m&ndash;RNA DNA hybrid no primer is required as the 3' end of this S-S C-DNA serves as its own primer generating a short hairpin 100p at this end.
This free 3' OH is required for the synthesis.
S1Nuclease produce blunt ends in C&ndash;DNA
Blunt end C-DNA termini are modified in order to ligate into a vector to prepare ds C-DNA for cloning. Since blunt end, ligation is inefficient short restriction site linkers are first ligated to both ends.
Linker - It is a double-strand DNA segment with a recognition site for a particular restriction enzyme. It is a 10&ndash;12 base pair long prepared by hybridizing chemically synthesized complementary oligonucleotides. The blunt-ended d.s. DNA is ligated with the linkers by the DNA ligase from Tu Bacteriophage.
The resulting double-strand C-DNA with linkers at both ends is treated with a restriction enzyme specific for the linker generating C-DNA molecule with sticky ends.
Problems arise when C-DNA itself has a site for the restriction enzyme cleaving the linkers.
This can be overcome using an appropriate modification enzyme (methylase) to protect any internal recognition site from digestion which methylase specific bases within the restriction site sequence thereby preventing the restriction enzyme binding.&nbsp;

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A DNA library is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments.

DNA Library - Types of DNA Library | Genomic Library & cDNA Library

A DNA library is a population of DNA fragments ready for sequencing. We can say DNA library is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments. Here we will discuss types of DNA Library, Genomic Library, Screening of the genomic library, cDNA Library, Differences between Genomic Library & cDNA Library and Isolation of mRNA. Read further for more details!

DNA Library - Types of DNA Library

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