CONSTRUCTION OF GENOMIC LIBRARY

Sources:
1. Biotechnology By B.D. Singh,
2. Gene cloning and DNA analysis By TA Brown,
3. Advance and Applied Biotechnology By Marian Patrie,
4. Molecular Biotechnology By Glick et al and
5. Principle of Gene Manipulation By sandy b Primrose et al.

Dr. Diptendu Sarkar

[email protected]
 Introduction
• A genomic library is an organism specific collection of DNA, covering the entire genome of an organism.
• A genomic library is a set of recombinant clones that contains all of the DNA present in an individual
organism.
• Genomic libraries can be retained for many years,and propagated so that copies can be sent from one
research group to another.
• It contains all DNA sequences such as :
1. expressed genes,
2. non-expressed genes,
3. exons and introns,
4. promoter and terminator regions and
5. intervening DNA sequences.

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 Construction of genomic library
• Construction of a genomic DNA library involves
o isolation,
o purification and fragmentation of
genomic DNA
o followed by cloning of the fragmented
DNA using suitable vectors.
• The eukaryotic cell nuclei are purified by
digestion with protease and organic (phenol-
chloroform) extraction.
• The derived genomic DNA is too large to
incorporate into a vector, and needs to be broken
up into desirable fragment sizes.
• Fragmentation of DNA can be achieved by
physical method and enzymatic method. Preparation of a gene
library in a cosmid
• The library created contains representative vector.
copies of all DNA fragments present within the
genome.

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 Mechanisms for cleaving DNA
(a) Physical method
It involves mechanical shearing of genomic DNA using a narrow-gauge syringe needle or sonication to break
up the DNA into suitable size fragments that can be cloned. Typically, an average DNA fragment size of about
20 kb is desirable for cloning into λ based vectors. DNA fragmentation is random, which may result in variable
sized DNA fragments. This method requires large quantities of DNA.

(b) Enzymatic method

 It involves use of restriction enzyme for the fragmentation of purified DNA.
 This method is limited by distribution probability of site prone to the action of restriction enzymes which
will generate shorter DNA fragments than the desired size.
 If, a gene to be cloned contains multiple recognition sites for a particular restriction enzyme, the complete
digestion will generate fragments that are generally too small to clone. As a consequence, the gene may
not be represented within a library.
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 To overcome this problem, partial digestion of the
DNA molecule is usually carried out using known
quantity of restriction enzyme to obtain fragments
of ideal size.
 The two factors which govern the selection of the
restriction enzymes are- type of ends (blunt or
sticky) generated by the enzyme action and
susceptibility of the enzyme to chemical
modification of bases like methylation which can
inhibit the enzyme activity.
 The fragments of desired size can be recovered by
either agarose gel electrophoresis or sucrose
The complete (a) and partial (b) digestion of a DNA fragment using
restriction enzymes.
gradient technique and ligated to suitable vectors.

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Partial restriction digestion is achieved using restriction enzymes that produce blunt or sticky ends as
described below-

i. Restriction enzymes generating blunt ends

The genomic DNA can be digested using restriction enzymes that generate blunt ends e.g. HaeIII and AluI.

Blunt ends are converted into sticky ends prior to cloning. These blunt ended DNA
fragments can be ligated to oligonucleotides, that contain the recognition sequence for a restriction enzyme,
called linkers or possess an overhanging sticky end for cloning into particular restriction sites called adaptors.

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Linkers
Linkers are short stretches of double stranded DNA of
length 8-14 bp that have recognition site for restriction
enzymes. Linkers are ligated to blunt end DNA by
ligase enzyme. The linker ligation is more efficient as
compared to blunt-end ligation of larger molecules. The
ligated DNA can be digested with appropriate
restriction enzyme generating cohesive ends required
for cloning in a vector. The restriction sites for the
enzyme used to generate cohesive ends, may be
present within the target DNA fragment which may
limit their use for cloning.
Linkers and their use: (a) the structure of a typical linker;
(b) the attachment of linkers to a blunt-ended molecule.

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Adapters

These are short stretches of oligonucleotide with

cohesive ends or a linker digested with restriction
enzymes prior to ligation. Addition of adaptors to
the ends of a DNA converts the blunt ends into
cohesive ends.

Adaptors and the potential problem with their use.

(a) A typical adaptor. (b) Two adaptors could ligate to one another
to produce a molecule similar to a linker, so that (c) after ligation
of adaptors a blunt-ended molecule is still blunt-ended and the
restriction step is still needed.

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 The use of adaptors: (a) the actual
structure of an adaptor, showing the
modified 5’-OH terminus; (b) conversion of
blunt ends to sticky ends through the
attachment of adaptors. After the adaptors
have been attached, the abnormal 5′-OH
terminus is converted to the natural 5′-P
form by treatment with the enzyme
polynucleotide kinase ,
producing a sticky-ended fragment that
can be inserted into an appropriate vector.

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ii. Restriction enzymes that generate sticky
ends
Genomic DNA can be digested with
commonly available restriction enzymes, that
generate sticky ends.
For example, digestion of genomic DNA with
the restriction enzyme Sau3AI (recognition
sequence 5’-GATC-3’) generates DNA
fragments, that are compatible with the sticky
end produced by BamHI (recognition
sequence 5’-GGATCC-3’) cleavage of a vector.
Once the DNA fragments are produced, they
are cloned into a suitable vector.
Construction of genomic library

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 Cloning of genomic DNA
Various vectors are available for cloning large DNA fragments. λ phage, yeast artificial chromosome, bacterial
artificial chromosome etc. are considered as suitable vectors for larger DNA and λ replacement vectors like
λDASH and EMBL3 are preferred for construction of genomic DNA library. T4 DNA ligase is used to ligate the
selected DNA sequence into the vector.

(1) λ replacement vectors

The λEMBL series of vectors are widely used for genomic library construction. The multiple cloning sites of
these vectors flanking the stuffer fragment contain opposed promoters for the T3 and T7 RNA polymerases.
The restriction digestion of the recombinant vector generates short fragments of insert DNA left attached to
these promoters. This generates RNA probes for the ends of the DNA insert. These vectors can be made
conveniently, directly from the vector, without recourse to sub-cloning.

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λ replacement vector
1.Preparation of arms and genomic
inserts

2.Ligation
3. Packing with a mixture of the phage coat
proteins and phage DNA-processing
enzymes

4. Infection and formation of

plaques

Library constructed
(2) High-capacity vectors

The high capacity cloning vectors used for the construction of genomic libraries are cosmids, bacterial artificial
chromosomes (BACs), P1-derived artificial chromosomes (PACs) and yeast artificial chromosomes (YACs).
They are designed to handle longer DNA inserts, much larger than for λ replacement vectors. So they require
lower number of recombinants to be screened for identification of a particular gene of interest.

The recombinant vectors and insert combinations are grown in E. coli such that a single bacterial colony or
viral plaque arises from the ligation of a single genomic DNA fragment into the vector.

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 Vectors used for cloning genomic libraries.
VECTOR INSERT SIZE FEATURES

1. λ phages Up to 20-30 kb Genome size-47 kb, efficient packaging system, replacement

vectors usually employed, used to study individual genes.
2. Cosmids Up to 40 kb Contains cos site of λ phage to allow packaging, propagate in E.
coli as plasmids, useful for sub-cloning of DNA inserts from YAC,
BAC, PAC etc.
3. Fosmids 35-45 kb Contains F plasmid origin of replication and λcos site, low copy
number, stable.
4. Bacterial artificial Up to 300kb Based on F- plasmid, relatively large and high capacity vectors.
chromosomes (BAC)
5. P1 artificial Up to 300 kb Derived from DNA of P1 bacteriophage, combines the features of
chromosomes (PACs) P1 and BACs, used to clone larger genes and in physical mapping,
chromosome walking as well as shotgun sequencing of complex
genomes.
6. Yeast artificial Up to 2000kb Allow identification of successful transformants (BAC clones are
chromosomes (YAC) highly stable and highly efficient)
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 Number of clones required for a library
• The number of clones to be pooled depends upon the size of the genome and average size of the cloned
DNA.
• Let f be the size of the genome relative to a single cloned fragment. The minimum number of clones
required can be calculated as : f= genome size/ fragment size
• For the E. coli genome (4.6 Mb) with an average cloned fragment size of 5 kb, f will be 920. Thus, for the
human genome (2.8 ×106 kb) and an average cloned fragment size of 20 kb, f= 1.4 × 105.
• The number of independent recombinants required in the library must be greater than f, as sampling
variation leads to the several times inclusion and exclusion of some sequences in a library of just f
recombinants.
• In 1976, Clarke and Carbon derived a formula to calculate probability (P) of including any DNA sequence in
a random library of N independent recombinants. The actual number of clones required can be calculated
as- [ N= ln (1-P) / ln (1-1/f) ] ; where N= number of clones and P= probability that a given gene will be
present.
Therefore, to achieve a 95% probability (P =0.95) of including any particular sequence in a random human genomic DNA
library of 20 kb fragment size: N= ln (1- 0.95) / ln (1- 1/ 1.4 × 105) = 4.2x 105.
• Notice that a considerably higher number of recombinants is required to achieve a 99% probability, for here N =6.5 ×105.
• Bigger the library better will be the chance of finding the gene of interest.
• The pooling together of either recombinant plaques or bacterial colonies generates a primary library.
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 Amplified library
• The primary library created is usually of a low titer and unstable. The stability and titer can be increased by
amplification. For this, the phages or bacterial colonies are plated out several times and the resulting
progenies are collected to form an amplified library.
• The amplified library can then be stored almost indefinitely due to long shelf-life of phages.
• It usually has a much larger volume than the primary library, and consequently may be screened several
times.
• It is possible that the amplification process will result in the composition of the amplified library not truly
reflecting the primary one.
• Certain DNA sequences may be relatively toxic to E. coli cells. As a consequence bacteria harboring such
clones will grow more slowly than other bacteria harboring non-toxic DNA sequences. Such problematic
DNA sequences present in the primary library may be lost or under-represented after the growth phase
required to produce the amplified library.
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 Sub genomic library

• Sub genomic library is a library which represents only a fraction of the genome.

• Enhancing the fold of purification of target DNA is crucial for sub genomic DNA libraries which can be

achieved by multiple, sequential digestion when information of the restriction map of the sequences of

interest is known.

• After initial purification of a given fragment, the purification can further be increased by redigestion with

another enzyme generating a smaller (clonable) fragment relative to original DNA.

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 Advantages of genomic libraries

• Identification of a clone encoding a particular gene of interest.

• It is useful for prokaryotic organisms having relatively small genomes.
• Genomic libraries from eukaryotic organisms are very important to study the genome sequence of a
particular gene, including its regulatory sequences and its pattern of introns and exons.

Disadvantages of genomic library

• Genome libraries from eukaryotes having very large genomes contain a lot of DNA which does not code
for proteins and also contain non-coding DNA such as repetitive DNA and regulatory regions which makes
them less than ideal.
• Genomic library from a eukaryotic organism will not work if the screening method requires the expression
of a gene.

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 Applications

1. To determine the complete genome sequence of a given organism.

2. To study and generate transgenic animals through genetic engineering, serving as a source of genomic
sequence.
3. To study the function of regulatory sequences in vitro.
4. To study the genetic mutations.
5. Used for genome mapping, sequencing and the assembly of clone contigs.

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