DNA libraries allow for the storage and organization of genetic information, similar to how physical libraries store books. There are two main types of DNA libraries: genomic libraries, which are created from genomic DNA and contain entire genes with exons and introns, and cDNA libraries, which are created from mRNA and contain only exons. To create a genomic library, genomic DNA is isolated, fragmented, and inserted into cloning vectors within host bacteria. For cDNA libraries, mRNA is isolated, reverse transcribed into cDNA, which is then amplified and inserted into vectors. Both library types are screened to find clones containing desired DNA sequences.

1. DNA Libraries
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Similar to Book collections can be stored in a library; collections of
genes can be made and stored in gene libraries !
1.Genomic libraries are made from DNA and contain entire genes
(exons and introns).
2.cDNA libraries are made from mRNAs that are converted into
DNA (only exons)
cDNA libraries are made from mRNAs
It contains only exons.
Libraries

3. Genomic libraries
A genomic library is a population of host bacteria, each of
which carries a DNA molecule that was inserted into a
cloning vector, such that the collection of cloned DNA
molecules represents the entire genome of the source
organism.
Genomic libraries are made from DNA and contain entire
genes (exons and introns).

4. Construction of Genomic library
1) Isolation of chromosomal DNA
2) Fragmentation of DNA:-
It is cleaved by 2 ways
a) Mechanical shearing - it involves pipetting, physical agitation
(vortexing), sonication.
b) Restriction enzyme digestion
Advantages:
It produce cohesive DNA ends for efficient ligation.
Choose enzymes for different average insert sizes.
(e.g. 4-base cutter, 6-base cutter, longer recognition site)
Disadvantages:
We may get fragment of different sizes, due to random
distribution of enzyme sites.
Much eukaryotic DNA is extensively methylated. Enzymes may
not recognize methylated DNA.
We can vary time of digestion (or of physical treatment) to
produce DNA fragments of desired size.

5. (a) Complete digestion ensures that
all restriction enzyme recognition
sites (RE) are cut.
(b) Partial digestion results in the
cleavage of a random subset of the
recognition sites. Partial digestion
will generate a variety of products as
indicated
Some RE are blunt end cutters e.g.
HaeIII and AluI.
The blunt ended DNA fragments are
problematic while its cloning because
the ligation of sticky-ended DNA is
more efficient than blunt ended DNA
fragment.

6.  Sticky ends DNA fragments are produced in 2 ways:-
1. Digestion with Restriction enzymes that generate sticky
ends.
2. Addition of Linkers or adaptors.

7. Linkers or adaptors:
The blunt ended DNA
fragments can be ligated
to oligonucleotides that
either contain the
recognition sequence for a
restriction enzyme
(linkers) or possess one
blunt end for ligation to
the genomic DNA and an
overhanging sticky end for
cloning into particular
restriction sites (adaptors).

8. 3) Isolation and ligation of
DNA fragments:-
Purify fragments of
required size by agarose
gel electrophoresis, and
elution of fragments
from gel.
Ligate fragments into a
vector which is pre-cut
with a restriction
enzyme, using DNA
ligase.

10. cDNA libraries
All cells within an individual
organism are derived from the
same genome sequence, but the
genes expressed in cell is unique to
individual cell type e.g. some of the
genes expressed within a skin cell
will be different to those of a
muscle cell.
DNA – all genes are present in
every cell
Only some genes are expressed in a
given cell
mRNA population represents those
genes expressed in a given cell
(tissue specific gene expression)
Thus, the mRNA of a cell gives us a
snapshot of the genes which are
expressed within that cell at any
particular time. Central Dogma of Molecular
Genetics

11. DNAExon1 Exon2 Exon3
IntronI IntronII
D
mRNA
Mature mRNA
D
Start
AAAAAAA polyA tail
Stop
Protein
Intron splicing and
polyA tailing
Translation on
ribosome
Open Reading Frame (ORF)

12. Need for making cDNA
The problem with mRNA is, that it cannot be maintained in
stable vectors and is difficult to manipulate.
DNA copy (called complementary DNA, or cDNA) of the mRNA is
required before a library can be constructed.

13. Reverse transcriptase
David Baltimore and Howard Temin first discovered the enzyme
in 1970
The conversion of RNA to DNA is dependent upon the action of
reverse transcriptase enzyme found in retroviruses.
Reverse transcriptase is an RNA-dependent DNA polymerase
that, like all other DNA polymerases, catalyses the addition of
new nucleotides to a growing chain in a 5 to 3 direction.
Reverse transcriptases generally have two types of enzymatic
activity.
1.DNA polymerase activity: reverse transcriptase produces a DNA
copy from RNA
2.RNaseH activity: RNaseH is a ribonuclease that degrades the RNA
from RNA-DNA hybrids,

14. Construction of cDNA
Isolation of mRNA –
• The presence of a poly-A tail is unique to mRNA, and provides a
mechanism of distinguishing and isolating mRNA from the more
rRNA and tRNA molecules.
• mRNA can be physically isolated by passing total RNA over a
column, to which polymers of deoxythymidine (oligo-dT) are
bound
• RNA molecules that do not contain multiple adenine residues
will be unable to adhere to such a column and will flow straight
through the column.
• mRNA molecules, will bind through complementary base
pairing to the column And will be eluted out by changing the
salt concentration.

15. The cloning of cDNA is initiated by
mixing short (12–18 base)
oligonucleotides of dT (oligo dT) with
purified mRNA.
The oligonucleotide will anneal to the
poly-A tail of the RNA molecule.
Reverse transcriptase is then added.
It uses the oligo-dT as a primer to
synthesize a single strand of cDNA in the
presence of the four deoxynucleotide
triphosphates (dNTPs).
The resulting molecules will be double-
stranded hybrids of one cDNA and one
mRNA molecule.
The primer can pair at numerous
positions throughout the polyA tail and
consequently will yield cDNA fragments
of different lengths which may have
been derived from the same mRNA
molecule.
First DNA strand formation

16. Besides priming the poly(A)
sequence at the 3 end of mRNA, the′
oligo(dT) primer can also prime the
poly(A) sequences present internally
within mRNA. The initiation of cDNA
synthesis from the oligo(dT) primed
at both locations results in the
generation of two truncated cDNAs
from a single mRNA template.
To overcome this problem, we use
anchored oligo-dT primers.
In addition to the 12–18 base dT
sequence, anchored primers are
constructed such that the extreme 3-
end contains either a G, A, or C
residue.
Such primers will initiate DNA
replication if they are paired at the
extreme 5-end of the polyA tail, when
the G, A, or C residue can base pair
with the nucleotide immediately
preceding the polyA sequence.

17. Second DNA strand formation
In early years, a hair-pin serve as a self primer for second stand.
The hair-pin would be removed from the double-stranded cDNA
by treatment with S1 nuclease. However, such methods
invariably resulted in the loss of sequences at the 5-end of
genes.
Now days the second DNA strand is synthesized in 2 ways:-
Nick translation
Homopolymer tailing.

18. • RNaseH is used to partially digest the RNA component of the
RNA–DNA hybrids.
• The remaining RNA is used as a primer for DNA synthesis using
DNA polymerase in the presence of the four dNTPs
• DNA ligase is used to seal nicks in the DNA backbone , resulting
double-stranded cDNA formation.
Nick translation

19. The RNA–DNA hybrids are treated with the enzyme terminal
transferase in the presence of a single deoxynucleotide
triphosphate.
Terminal transferase catalyse the addition of deoxynucleotides
to the 3-ends of DNA.
Homopolymer tailing

20. The RNA of the RNA–DNA
hybrids must be removed to
provide a single-stranded
template for new DNA
synthesis.
This is achieved by treating
the hybrids with alkali.
RNA is hydrolysed into
ribonucleotides around pH 11,
while DNA is resistant to
hydrolysis up to about pH 13
Increasing the pH to about 12
results in the hydrolysis of the
RNA, but not the DNA.

21. Second-strand cDNA
synthesis is then
initiated using an oligo-
dG primer that will bind,
through complementary
base pairing, to the
newly formed polyC
sequence.
Reverse transcriptase in
the presence of the four
dNTPs will produce the
second cDNA strand.

24. Vector type Cloned DNA (kb)
Plasmid 20
lambda phage 25
Cosmid 45
BAC (bacterial artificial chromosome) 300
YAC (yeast artificial chromosome
1000
Vector systems

25. 1.Screening by Nucleic Acid
Hybridization:
The main advantage of this type
of screening is that it can be
applied to almost any vector
system.
The colonies to be screened are
grown on agar plates that
contain the appropriate
antibiotics that allows the
growth of recombinant
molecules.
Nitrocellulose membrane is
placed on top of them and then
lifted off to produce a replica
version of the plate.
The nylon replica is treated to
lyse the bacteria and firmly
attach the DNA to the sheet.
Screening of recombinants

26. Steps:-
1. The nylon sheet is treated with alkali (e.g. 0.5 M NaOH) to
initiate both bacterial cell lysis and DNA denaturation.
2. Upon neutralization, the sheet is treated with proteases (e.g.
proteinase K) in order to remove the protein and leave the
denatured DNA bound to the membrane.
3. The sheet is then baked at 80 C, or treated with UV light, to◦
firmly adhere the DNA to the membrane.
4. Now nitrocellulose sheet containing a denatured DNA copy of
the bacterial colonies originally present on the agar plate.
Hybridize the DNA on the nitrocellulose membrane with a
labelled probe.
It locates the colonies by radioactivity of probe, on the original
dish that contain identical DNA sequences.

27. Probe designing
Purified biochemically.
Digested with the enzymes.
The resulting protein
fragments were subjected
to amino acid sequencing
All possible DNA sequences
that could encode this
peptide were determined
Antisense DNA probe are
constructed.

28. Immunoscreening
cDNA is cloned into the expression vector under the control of
the bacterial lac promoter.
Recombinant λ phages are plated out onto a suitable bacterial
host on agar plates.
Incubated until small plaques appear.
Place a nitrocellulose sheet soaked in IPTG on top of the
plaques.
It bind the proteins produced by the E. coli cells after lyse by
the phage infection.
Peel off the nitrocellulose membrane
Incubate membrane with a specific antibody (against protein
product of gene which we clone).
Incubated membrane with a labelled secondary antibody to
detect the presence of the bound primary antibody.

30. Screening by Function
Host cell is required that either lacks a biochemical function.
Those cell are screened as recombinants that are able grow.
For example,
Select the E. coli cells that are defective in imidazole glycerol
phosphate dehydratase (essential for the biosynthesis of
histidine) - unable to grow on media lacking histidine.
 If the hisB defective E. coli cells are transformed with an
expression library from yeast and plated onto media lacking
histidine, the only recombinant cell are able to grow.
The yeast HIS3 and E. coli hisB genes share little DNA sequence
similarity (less than 20% ), but the encoded proteins, perform
the same enzymatic function.

31. Thanks
Acknowledgement: All the material/presentations available online on the subject
are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
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