A series of books in a library helps to study a suitable topic in detail. The detailed research and analysis of a topic require a lot of information. Different books cover a particular topic with different findings. Hence, the research studies involve reference to several books discussing a topic in different aspects. Hence, a book library is nothing but a collection of books discussing different subjects. Just like a book library, the molecular world depends on the DNA libraries. Recombinant DNA libraries consist of a collection of clones of individual DNA fragments, complementary DNA (cDNA) fragments, and chromosome fragments. Hence, they consist of three main types of libraries such as genomic libraries, cDNA libraries, and chromosome libraries. Recombinant DNA libraries help the researchers study a particular gene segment, DNA or a chromosomal segment isolated from any organism. It is possible to isolate a specific gene and study its structure, function, and expression using recombinant DNA libraries.
Clone libraries involve a collection of clones for generating mapping reagent. A collection of clones represent an entire genome. Hence, they supply individual clones of interest. Clone libraries contribute in physical and gene mapping techniques as well. Preparation of a clone library either involves a genomic DNA or a specific chromosome.
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| Image: Recombinant vectors |
Genomic library:
They consist of at least one copy of every DNA sequence in the genome. It is possible to isolate and study a particular gene using a genomic library. The first step of constructing a genomic library involves digesting the DNA sample with restriction enzymes. Next step follows cloning the DNA fragment obtained from restriction digestion into a vector. The vector consists of a specific restriction site. First, the restriction site in the vector undergoes cleavage due to the action of a specific restriction endonuclease. The vector now has a space for new DNA insert. Upon treating the vector with new DNA fragment and specific ligating enzymes, the DNA gets inserted into the vector. Hence, the genomic library gets prepared. However, a limitation exists to the above method. Suppose, if a gene consists of one or more restriction sites for a specific restriction enzyme, it gets cleaved into two or more fragments smaller in sizes. In this way, it produces a large number of fragments. Cloning the longer DNA fragments into a vector helps in solving the problem. Mechanical shearing gives longer DNA fragments. Another approach involves partial digestion. The restriction enzymes recognize four to six base pair nucleotide sequences. Partial digestion results in the formation of overlapping fragments.
Uses of genomic libraries:
1. Genomic libraries help in the sequencing projects such as whole genome sequencing, human genome sequencing, and sequencing related to another organism.
2. It is possible to create many recombinant DNA molecules using a genomic library.
3. A genomic library has a larger size. It consists of an entire genome of an organism.
4. The genomic library helps in molecular cloning of a gene.
The cDNA libraries:
The mRNA molecules help in deriving the cDNA (complementary DNA). Cloning the cDNA molecules involve a contribution of a suitable vector for producing cDNA library. Most f the mRNA molecule consists of a poly(A) tail. Polyadenylation process occurs after the mRNA synthesis and capping. The poly (A) tail with mRNA gets purified with the help of deoxythymidylic acid known as oligo (dT) chain. The poly (A) tails base pair with the oligo (dT) chains. The mRNA gets captured on the column leaving the remaining molecules. After annealing the oligo (dT) primer to a poly (A) tail, the primer gets extended using a reverse transcriptase enzyme. This enzyme helps in making a DNA copy of the mRNA strand. The second DNA fragment gets synthesized with the help of RNase H, DNA polymerase I, and DNA ligase. The DNA polymerase I synthesize a new DNA strand and removes the primers. Then the ligating enzyme joins them. The result is a double-stranded DNA which is known as cDNA or a complementary DNA.
Cloning of a cDNA involves a restriction site linker. It is a short, double-stranded piece of DNA with 8-12 nucleotide pairs in length. The linker has blunt ends. Hence, cDNA gets ligated with a linker (such as Bam HI) using a T4 ligase enzyme. Now the linkers get cleaved using a Bam HI restriction enzyme. The restriction enzyme cleaves the linker at a particular nucleotide resulting in sticky ends. The resultant product gets inserted into a vector cleaved with Bam HI. The recombinant vector gets transformed into a host such as E coli. There is a drawback of using a linker. The cDNA may have restriction sites just like the linker DNA. Hence, there are chances of cDNA getting cleaved with a restriction enzyme. It is required to prevent the cDNA digestion with the restriction enzymes. An adapter helps in doing so. Upon ligation, the blunt end of the adapter covalently attaches to the blunt end of the cDNA. It leaves a 5’ overhang at each end capable of base pairing with a vector.
Uses of a cDNA library:
1. The cDNA libraries are small and precise. They do not involve the non-coding regions or the introns. Hence, they contribute to studying the coding regions or exons.
2. They express eukaryotic genes in prokaryotes.
3. Helps to study reverse genetics.
Chromosome libraries:
Libraries for specific human chromosomes are also available. Also, the artificial chromosome libraries are available. Examples include BACs and YACs. The Bacterial artificial chromosome (BACs) libraries consist of bacterial chromosomes or DNA cloned into vectors. YACs are also known as yeast artificial chromosomes and derived from yeast.
Deriving a human chromosome library involves separating the chromosomes by flow cytometry. The first step involves carefully breaking apart the dividing cells for obtaining the chromosomes. The next step involves staining the chromosomes with a fluorescent dye. The larger chromosomes require more dye and fluoresce more brightly. Chromosome libraries serve in STS mapping. We use chromosome library as a mapping reagent or obtain a DNA sequence using overlapping clones.
References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu
[2] Recombinant DNA Principles and Methodologies, James Greene
[3] Gene Cloning and Manipulation, Christopher Howe
[4] Genetic Engineering, Verma P.S. & Agarwal V.K.
[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental, S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown
[6] Gene cloning and DNA analysis, T.A. Brown
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