8. DNA LIBRARY
8.1. Genomic Library
The genomic library represents as 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.
Construction of a genomic library
DNA isolation: isolate complete DNA from any cell under study.
Cutting isolated DNA with restriction fragments of suitable size: Restriction enzymes like Eco RI are used to cut into fragment into a suitable size.
Step 3 Incorporation of this fragment to a suitable vector like a plasmid, cosmid etc leading to the formation of the rDNA molecule.
Introduction of rDNA molecule into a suitable host as E.coli. The plasmid multiplies inside the host forming numerous copies.
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 library are plated out on a Petri dish and allow 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 & placed in a sealed bag with the labelled 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.
The advantage of Genomic Library
Identification of a clone encoding a particular gene of interest. It is useful for prokaryotic organism having relatively small genomes. Genomic library from the eukaryotic organism is 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
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–p) / ln (1 – 1/f)
Where N = number of clones
P = Probability that a given gene will be present.
8.2. cDNA Library
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.
8.3. Differences between Genomic Library & cDNA Library
8.4. Isolation of mRNA
In eukaryotes, gene expression is tissue specific for ex. the gene encoding globin proteins are expressed only in erythrocyte precursor cell called reticulocytes using this information a target gene can be cloned by isolating the m-RNA from a specific tissue.
m–RNA can be isolated by
Spinning down m-RNA by density gradient centrifugation.
The 3' end of m-RNA consist of 50-250 adenylate residue (Poly-Atail) so it can separate easily by oligo dT on the resin.
A complementary DNA is synthesized from m–RNA with the help of RTc.
RTc has three special feature of catalys.
1. RNA dependent DNA polymerase
3. DNA dependent DNA polymerase.
This RTc obtains from avian myeloblastosis virus.
RNase–H removes – mRNA from m–RNA DNA hybrid no primer is required as the 3' end of this S-S C-DNA serve as its own primer generating a short hairpin 100p at this end.
This free 3' OH is required for the synthesis.
S1 Nuclease produce blunt ends in C–DNA
Blunt end C-DNA termini have modified the 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 10–12 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 are 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 (a methylase) to protect any internal recognition site from digestion which methylase specific bases within the restriction site sequence thereby preventing the restriction enzyme binding.
- TOOL AND TECHNOLOGY
- HYBRID PLASMID / PHAGE VECTORS
- ARTIFICIAL CHROMOSOMES
- SHUTTLE VECTORS
- ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY
- DNA LIBRARY
- FLUROSCENT ACTIVATED CELL SORTER
- DNA MICROARRAY OR GENE CHIP OR BIO CHIP
- ANTIBODY GENERATION
- RADIOIMMUNOASSAY (RIA)
- ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY
- POLYMERASE CHAIN REACTION
- TYPE OF HYDROLYSIS PROBE
- X-RAY DIFFRACTION
- NMR (NUCLEAR MAGNETIC RESONANCE)
- CIRCULAR DICHROISM
- DNA SEQUENCING
- TRANSGENIC ANIMALS
- CRE–LOX P RECOMBINANT SYSTEM
- GENE THERAPY
- TRANSGENIC PLANTS
- PLANT TISSUE CULTURE (PTC)
- MICRO PROPAGATION
- ARTIFICIAL SEEDS
- PRACTICAL APPLICATIONS OF PLANT TISSUE CULTURE
- ANIMAL CELL CULTURE