Genomic Library

• A gene library is a collection of different DNA sequences from an organism, which has been cloned into vectors for ease of purification, storage, and analysis
• There are two types of gene libraries based on the availability of a source of DNA used. 
• The genomic library contains DNA fragments that represent the entire genome of an organism, whereas cDNA libraries will contain only the complementary DNA molecules synthesized from the mRNA molecules in a cell. 
• For the construction of gene libraries, we have to know the size of the gene and insert size or capacity of vectors

1.0What is a Genome?

• A genome is the complete set of genetic information in an organism. It provides all of the information the organism requires to function
• Total number of genes present in a haploid cell of an organism.
• The human genome has 23 chromosomes and 3 billion nucleotide pairs.

2.0Creation of Genomic Library

It has the following steps:

1. Isolation and purification of DNA

2. Digestion of DNA with RE

3. Separation of the gene of interest

4. Formation of rDNA

5. Gene transfer in a suitable host

6. Selection of recombinant transformant host

7. Cultured in medium

8. Screening of clones

3.0Procedure of Genomic Library Creation

• DNA is isolated from cells by using digestive enzymes like lysozyme, chitinase, protease, lipase, cellulase etc.
• Isolated DNA is separated by using chilled ethanol through a spooling method.
• Isolated DNA is digested by using restriction endonuclease enzymes.
• Digested DNA fragments run into agarose gel so fragments arranged in descending order of its molecular weight towards the anode, the desired gene is separated from gel.
• Vector is cut with the same restriction endonuclease which was used for cutting DNA fragments. Vectors are used according to the size of the desired gene.
• Some examples of vectors and their capacity to carry foreign gene is as follows;

• Desired genes are ligated with vector DNA with the help of a ligase enzyme, this combination is known as recombinant DNA or rDNA.
• Recombinant DNA is transferred in suitable host and recombinant transformant host selected with the help of selectable markers of vector DNA.
• A heterogeneous host is cultured in the medium and transferred for screening of clones.

4.0Screening of Genomic library

• Library screening is used to identify the genes of interest. The two most common screening methods are colony hybridization and plaque hybridization, and the method which is chosen  will depend on the cloning vector you used.
• Colony hybridization is a method of selecting bacterial colonies containing the desired genes. This method is used to identify genes in a plasmid- or cosmid-based library. 
• Plaque hybridization on the other hand is used to identify the desired genes in phage-based libraries. Both methods use filter hybridization, enabling the primary screening of libraries by in situ replication on a nitrocellulose membrane.

5.0Advantages and Disadvantages of Genomic Library

Advantages:

• Genomic libraries are suitable for a wide range of applications, including genome mapping and comparative genomics. It allows the study of regulatory elements and noncoding sequences that are important in gene expression and regulation.
• Genomic libraries play an important role in DNA sequencing projects. It also plays an important role in identifying novel pharmaceutically important genes.
• The identification of new genes that are not active in the host is greatly facilitated by genomic libraries and enhances our understanding of the complexity of the genome.
• A major role in transgenic animal production is through the genomic sequence.
  

Disadvantages:

• The mishap in the construction of the gene library.
• Errors can occur in genetic maps. 
• Reference genome may be incomplete.

6.0Construction of cDNA Library

• The construction of cDNA libraries is achieved by the extraction of mRNA.
• This mRNA was extracted and purified by triazole extraction and column purification. 
• The mRNA is elated by using an elating buffer and some heat to separate the mRNA strands.
• Then, the mRNA is removed using RNase enzyme, leaving it as single-stranded cDNA.
• Once the cDNA is obtained, the same steps followed for the construction of the genomic library are followed.

Screening of cDNA Library: 

The identification of a specific DNA fragment allows for its isolation, subsequent amplification, and determination of its sequence. It is based on denature hybridization with a probe by performing autoradiography. 

Application of cDNA Library:

• cDNA library is used to isolate homogeneous genes. It helps express eukaryotic genes in prokaryotes.
• The major application includes studying the expression of mRNA. It can also facilitate the generation of antibodies and monoclonal antibodies. It plays an important role in the discovery of novel genes. To elucidate gene function, to get high yields of recombinant cDNA.
• Commercial applications are the production of proteins and other biological molecules. Various applications include identifying carcinogens, studying alternative splicing, and obtaining pure samples of genes

Disadvantages of cDNA Library: 

• Contains only sequences that are present in mRNA. 
• Introns and other sequences are altered after transcription is not present.
• Frequency of a particular DNA sequence in a cDNA library depends on the abundance of the corresponding mRNA.