Authors: Ghadage Nitin Chitrasen
Gene Library
A gene library is a collection of different DNA sequences from an organism, which has been cloned into a vector for ease of purification, storage and analysis. Gene library is a collection of cloned DNA designed so that there is a high probability of finding any particular piece of the source DNA in the collection. Gene libraries, like conventional libraries, are used to collect and store information as a set of DNA molecules. All Gene libraries are collections of DNA fragments that represent a particular biological system of interest.
There are two types of gene library that can be made depending upon the source of the DNA used.
- Genomic library
- cDNA library
To study anyone of these genes, a researcher first isolates it from all of the other genes in an organisms DNA. Researchers can identify and isolate the DNA fragments that interest them for further study.
When a gene is identified and copied, it is said to have been “cloned”
Types of Gene Library:-
- Genomic library :- Contains DNA fragments representing the entire genome of an organism.
A) Nuclear Genomic Library
B) Organelle Genomic Library -
cDNA library :- Contains only complementary DNA molecules synthesized from mRNA molecules in a cell.
- Genomic Library:- Are made from total nuclear DNA of an organism or species. DNA is cut into clonable size pieces as randomly possible using restriction endonuclease Genomic libraries contain whole genomic fragments including gene exons and introns, gene promoters, intragenic DNA, origins of replication, etc.
Construction of Genomic Library:- There are following main steps in gene cloning: 1. Isolation of genomic DNA and vector. 2. Cleavage of Genomic DNA and vector by Restriction Endonucleases. 3. Ligation of fragmented DNA with the vector. 4. Transformation of r-DNA in the bacterial cell. 5. Amplification of the r-DNA in bacterial cells.
- Screening of genomic library:- Once the genomic library has been created, it is screened to identify the genes of interest. One of the most common library screening technique is called colony hybridization. In the process of library construction, phage vectors are used then the process of identification of genes of interest involved is the plaques hybridization.
- Colony Hybridization:- Colony Hybridization is the screening of a library with a labeled probe (radioactive, etc.) to identify a specific sequence of DNA, RNA, enzyme, protein, or antibody.
- Plaque Hybridization:- The plaques are screened by a technique based on the hybridization of oligonucleotide probe to target DNA. In this case, DNA is transferred directly from the Petri dish to the filter, which is then incubated with labeled probes.
- Applications of Genomic Library:-
1. Genomic library construction is the first step in any DNA sequencing projects. 2. Genomic library helps in identification of the novel pharmaceutically important genes. 3. Genomic library helps in identification of new genes which were silent in the host. 4. It helps us in understanding the complexity of genomes. 5. Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering. 6. Study of the function of regulatory sequences in vitro. 8. Study of genetic mutations in cancer tissues. 9. Create cDNA libraries to determine what genes are being expressed at a particular time. - cDNA Library cDNA library is a collection of clones containing cDNA and represents the genes that are expressed within a given cell or tissue type at a particular period. cDNA represents collection of mRNA complements of single type of cell. cDNA libraries represent sequences after any post transcriptional modification (e.g. Splicing of introns). The advantage of cDNA library is that it contains only the coding region of a genome.
- Construction of cDNA Library
- Isolation of mRNA from cell & synthesis of cDNA from this eukaryotic functional mRNA
- Cloning of synthesized cDNAs in suitable vector like plasmid or ƛ phage.
- Introduction to suitable host like bacterium
- Screening & maintenance of set of clones containing cDNA
- Applications of cDNA Library
- Storage of reduced amount of information due to the removal of non-coding regions.
- cDNA can be directly expressed in prokaryotic organisms.
- cDNA libraries are useful in reverse genetics where the additional genomic information is of less use.
- cDNA library is useful for isolating gene that codes for particular mRNA.
- cDNA library is a powerful and useful tool in the area of biotechnology.
- It is helpful in expressing eukaryotic genes in prokaryotes, which helps in the transcription process of prokaryotes.
- It is used to isolate DNA sequences to code mRNA.
- Advantage of cDNA library is to isolate homologous genes.
- It is also used for the screening genomic libraries to isolate specific cDNA.
- cDNA of proteins can facilitate to generate antibodies and monoclonal antibodies.
- The most important application of cDNA library is to study expression of mRNA.
- Disadvantages of cDNA Library
- The disadvantage of a cDNA library is that it contains only sequences that are present in mature mRNA.
- Introns and any other sequences that are altered after transcription are not present; sequences, such as promoters and enhancers that are not transcribed into RNA also are not present in a cDNA library.
- It is also important to note that the cDNA library represents only those gene sequences expressed in the tissue from which the RNA was isolated.
- Furthermore, the frequency of a particular DNA sequence in a cDNA library depends on the abundance of the corresponding mRNA in the given tissue.
- Genomic Library Vs. cDNA Library
| Sr. No. | Genomic Library | cDNA Library |
| 1 | It include all possible fragments of DNA from a given cell or organism. | cDNA library carries only expressed gene sequences. |
| 2 | It is larger | It is smaller |
| 3 | It represents the entire genome of an organism having both coding and non-coding regions. | It represents only the expressed part of the genome and contain only coding sequences called ESTs |
| 4 | Expression of genes taken from genomic library is difficult in prokaryotic system like bacteria due to absence of splicing mechanism | cDNA has only coding sequences therefore can be directly expressed in prokaryotic system. |
| 5 | Vectors used genomic library include plasmid, cosmid, lambda phage, BAC and YAC in order to accommodate large fragments. | Vectors used cDNA library include plasmid, phagmids, lambda phage etc to accommodate small fragments as cDNA has no introns. |
- Storage of Gene Library
Therefore, it is necessary to store it safely for future use. The libraries are stored at – 80°C.
Bacterial cells in a plasmid library are protected from the adverse effects of freezing by glycerol, while phage libraries are cryoprotected by Di-Methyl Sulfoxide (DMSO).
References:
http://www.biologyexams4u.com/2017/05/cdna-library-definition-and-steps- notes.html#.WjNFT9KWZdh
2. www.springer.com/in/book/9781617790645
3. Fundamentals of Genetics by B.D Sing Pages 500-502
4.https://passel.unl.edu/pages/informationmodule.php?idinformationmodule=959197140&topi corder=15&maxto=16
About Author / Additional Info:
I am currently pursuing M.Sc (Agri) in Genetics & Plant Breeding from Junagadh Agricultural University, Junagadh, Gujarat 362001