DNA LIBRARY

The term "library" can refer to a population of organism, each of which

carries a DNA molecule inserted into a cloning vector, or alternatively to
the collection of all of the cloned vector molecules.
Collection of DNA fragments that have been cloned into vectors so that
researchers can identify and isolate the DNA fragments that interest
them for further study. For ease of purification, storage and analysis.
Two types of DNA Library

Genomic library
(made from genomic DNA)
DNA Library
CDNA Library
(made from cDNA- copy of mRNA)
For the construction of DNA Library
• Size of the gene
• Capacity of the vector
• Molecular tools
• Vectors
 Size of the Library
(ensure enough clones)
Must contain a certain no: of recombinants for there to be a high
probability of it containing any particular sequence.
The formula to calculate the no: of recombinants:
In (1-P)
N=-----------------------------------------
In (1-F)
P: desired probability
F: the fraction of the genome in one insert
Vectors for DNA Library

Vector Type Max. Insert size cloned DNA Approx.no: of cloned DNA
(kb) required in a library
Plasmid 20 >10^5

Lambda phage 20 5x10^5

Cosmid 45 2x10^5

BAC >500 5x10^4

YAC 1mb 10^5

What is Genomic Library?
• Contains DNA fragments representing entire genome of an organism.
• Created using molecular cloning
• Genome size is expressed in terms of no: of base pairs.
• The sizes of genomes in different species are variable. There is a
distinct difference in the genes of prokaryotes and eukaryotes.
• In prokaryotes, the structural genes coding for proteins are
continuous while in eukaryotes, the coding regions (exons) are
separated by non-coding regions (introns).
• Therefore, the construction of gene libraries for eukaryotes is more
complicated.
Steps of Genomic Library construction
• Isolation of DNA from cells
• Digestion into small fragments Introduction into suitable vectors
• Insertion into bacteria
• DNA isolation
• Collection of Genomic DNA library
 Isolation of DNA (purification)
• Eukaryotes: Prepare cell nuclei, remove proteins, lipids and other
unwanted macromolecules by protease digestion and phase
extraction.
• Prokaryotes: Extracted DNA directly from cells.
 Digestion into small fragments
Physical shearing: Pipetting, mixing
Restriction enzyme digestion: Partial digestion is preferred to get a
greater length of DNA fragments.
• DNA fragment to be cloned
 Selection of restriction enzymes
1. Ends produced (sticky or blunt) & the cleaved ends of the vector to
be vector
2. Whether the enzyme is inhibited by DNA modifications
3. Time of digestion and ratio of restriction enzyme to DNA is
dependent on the desired insert size range.
3. Introduction into suitable vectors
• Each fragment is different and have a unique DNA sequence
• Inserted into suitable vectors including plasmids and bacteriophage
vectors.
• Vectors are digested with the same Restriction Enzymes and sealed to
Human DNA using DNA Ligase enzyme.
• The resulting molecules are recombinant
•

4. Insertion into Bacteria

• Inserted into host bacteria (E.coli)
• The microbes are grown in culture.
• They are made to take up the DNA.
• They replicate their genome along with the vector genome contained
with them.
• Produce clones of the original genome.
• This collection of clones which contains all the sequences found in the
original source, including the sequence of interest forms the genomic
library.
What are the uses of Genomic Library?
• Researchers can explore the genome of an organism to learn more
about genomic structure and function
• They can map the genome, identifying the locations of specific genes.
• Helps to develop tests which can be used to locate genetic variations
including mutations
• Useful in Recombinant DNA Technology, helps to genetically modify
organisms and produce clones of desired types.
• Genomic library construction is the first step in any DNA sequencing
projects.
• Genomic library helps in identification of the novel pharmaceutically
important genes.
Applications of Genomic Library
• Genomic library is commonly used for DNA sequencing applications.
• They have played an important role in the whole genome sequencing
of several organisms including, the
• human genome and several model organisms. In case of E. coli, about
1500 DNA fragments are required to form genomic library, wheras 10
lakh DNA fragments are required for human DNA.
2. cDNA Library
cDNA library is a collection of only those genes that are expressed in a
given organism. mRNAs from an organism are extracted and
complementary double stranded DNAS (cDNAs) are produced by
reverse transcriptase. The resulting DNA fragments are inserted into
suitable vectors and cloned. The collection of these cloned DNA
fragments of the only genes that are expressed in an organism is called
cDNA library.
Steps
i. Isolation and Separation of Desired mRNA
The desired mRNA is isolated from the cells/tissue by various techniques.
ii. Reverse Transcription
• Reverse transcription is RNA-directed DNA synthesis. Reverse transcription
takes place when isolated mRNA is treated with oligo-dT primer in the
presence of reverse transcriptase enzyme along with all dNTPs (dATP, dGTP,
dCTP, dTTP). The oligo-dT primer binds to polyadenyl tail which provides free
hydroxyl site for reverse transcription. Reverse transcriptase adds
complementary dNTPs one by one to 3'-OH end of mRNA resulting in RNA-
DNA hybrid.
iii. Loop Formation
• When the RNA-DNA hybrid is treated with terminal transferase and nucleotide
precursors, formation of a short oligo-dC tail (curve tail) takes place. This curve
tail forms a loop-like structure.
iv. Alkali Treatment
• RNA-DNA hybrid so formed are separated by alkali treatment to form single
stranded cDNA.
v. Polymerization
• Now this cDNA act as a template for the synthesis of DS DNA in the presence of
DNA polymerase along with dNTPs. The polymerase adds the complementary
nucleotides to the existing cDNA resulting in the formation of double stranded
DNA.
vi. Breakage of Loop at C-C Terminal End
• Breakage of loop at C-C terminal end is catalysed by single strand specific nuclease
to form an active double stranded DNA (clone) which forms the basis of cloning.
vii. Cloning
• The clone is inserted into a suitable host through a vector to form a recombinant
DNA. This recombinant DNA is cultured in suitable media to obtain multiple copies
of the original clone.
Applications
• cDNA Library is used to express eukaryotic DNA in prokaryotes. It is
most useful in the study of reverse genetics (comparison of genomic
information of an advance organism to primitive one).