GENETIC ENGINEERING

UNIT II
DNA LIBRARY (GENETIC LIBRARY)
DNA Library or Gene Library is simply the collection of DNA fragments cloned
into vectors and stored within host organisms. They contain either the entire genome of a
particular organism or the genes that are expressed at a given time.
The genome is vast and complex. To understand the entire genome or study specific genes, it is
important to study it in smaller and more manageable fragments. DNA libraries make the entire
genome accessible in small fragments. These libraries are used to identify, isolate, and study
particular genes of interest.
The development of molecular biology technologies including recombinant DNA (rDNA)
technology, cloning vectors, and techniques for transforming bacteria laid the foundation for the
construction of DNA libraries.
Types of DNA Library
Genomic information can be obtained by two primary methods. Based on this, DNA libraries are
divided into two types: genomic and cDNA library.
1. Genomic Library
 A genomic DNA library is a collection of DNA fragments that represent all genetic
information of an organism. This includes both coding and noncoding regions of the DNA.
 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 also have certain disadvantages including the complexity and size of
handling large DNA fragments and the resource-intensive process of creating and
maintaining such libraries.
Construction (Preparation) of Genomic Library
1. Isolation of Genomic DNA
 The construction of a genomic library begins with the isolation of genomic DNA from the
organism of interest.
 Genomic DNA can be isolated using different methods such as cell lysis, protein digestion,
and phenol-chloroform extraction.
 The isolated DNA represents the entire genome of the organism and contains both coding
and non-coding regions.
2. Fragmentation of Genomic DNA
 The isolated genomic DNA obtained is often too large to be cloned directly into vectors.
So, it needs to be fragmented into smaller fragments suitable for cloning.
 This fragmentation can be achieved using physical methods such as sonication, mechanical
shearing, or enzymatic methods involving restriction enzymes.
3. Cloning
 The fragmented genomic DNA is then cloned into a suitable vector. Some common vectors
used for genomic library construction include plasmids, bacteriophages, bacterial artificial
chromosomes (BACs), and yeast artificial chromosomes (YACs).
 Bacterial vectors are suitable for smaller DNA fragments, while YACs are used for larger
DNA fragments.
 The vectors are treated with restriction enzymes to create sticky ends that are compatible
with the fragmented DNA.
 DNA ligase enzyme is used to bind the DNA fragments to the vector, creating recombinant
DNA molecules.
4. Transformation
 The recombinant vectors containing the cloned genomic DNA fragments are transformed
into a suitable host organism, usually E. coli and yeast.
 The transformed host cells take up the recombinant vectors and are cultured on agar plates
containing selective media to allow the growth of colonies containing the recombinant
DNA. These colonies form a genomic DNA library.

Applications of genomic libraries

 Genomic libraries are essential for constructing physical maps of genomes which helps in
understanding the layout and structure of genes.
 These libraries are particularly useful for studying non-coding regions, regulatory
elements, and gene sequences that may not be expressed.
 Genomic libraries from different species can be compared to study their evolutionary
relationships.
 Genomic libraries also help identify genetic mutations and disease-associated genes which
is important for understanding the genetic basis of diseases.
Construction (Preparation) of cDNA Library
1. Isolation of mRNA
 Construction of a cDNA library starts with the isolation of mRNA from eukaryotic cells.
 mRNA is isolated and purified using methods such as column purification. Column
purification uses oligomeric dT nucleotide-coated resins that bind only mRNA with a poly-
A tail.
 Eukaryotic mRNA contains a poly-A tail at the 3’ end. When a cell lysate is passed through
the poly-T column, the poly-A tails of mRNA molecules bind to the oligo-dT sequences.
 This retains the mRNA in the column while all other molecules that do not have poly-A
tails pass through the column and are discarded. Finally, the mRNA molecules are
separated from the oligo-dT sequences using an eluting buffer.
2. Synthesis of cDNA
 After isolating and purifying mRNA molecules, the next step is the synthesis of cDNA
molecules from the isolated mRNA.
 At first, a short oligo-dT primer is annealed to the 3’ poly-A tails of the mRNA molecule,
which initiates the synthesis of the first DNA. Using reverse transcriptase enzyme, the
primer is extended to form an RNA-DNA duplex.
 RNase H enzyme degrades the mRNA strand in the mRNA-DNA hybrid, leaving small
RNA fragments that are used as primers for the synthesis of the second DNA strand.
 DNA polymerase I synthesizes the second DNA strand in segments removing the RNA
primers.
 DNA ligase enzyme seals the nicks between the newly synthesized DNA fragments,
resulting in the formation of a double-stranded cDNA copy of the mRNA.
3. cDNA Cloning
 The next step is the ligation of cDNA molecules into suitable vectors for cloning.
 Since cDNA has blunt ends, restriction site linkers or adapters need to be added to the ends
of the cDNA molecules to make them compatible with the vector DNA. These linkers
contain recognition sites for restriction enzymes. The linkers are then digested using
restriction enzymes.
 The most commonly used vectors for cloning cDNA are plasmid and phage vectors. The
suitable vectors are cut with the same restriction enzyme as the cDNA.
 The cDNA molecules are joined with the vector DNA which creates recombinant DNA
molecules.
4. Transformation
 The recombinant DNA molecules are transformed into host cells that can be cultured to
produce colonies containing the cloned cDNA inserts.
 To select host cells that have successfully taken up the recombinant DNA, the transformed
cells are cultured on agar plates containing a selective medium. The selective medium
contains antibiotics that inhibit the growth of untransformed cells.
 The presence of a selectable marker gene on the vector ensures that only cells containing
the recombinant DNA survive and form colonies on the selective medium. The resulting
colonies form the cDNA library.

Applications of cDNA libraries

 cDNA libraries are useful for studying actively expressed genes in different tissues under
specific conditions. cDNA library allows the identification and cloning of expressed genes.
 cDNA libraries can be used to compare gene expression profiles between different species
which is useful in the study of evolutionary biology.
 cDNA libraries are also used to produce recombinant proteins.
  These libraries are also used to identify and study the expression of disease-related genes,
which can help in the development of diagnostic markers.

Screening of the clones from a gene library

A library has to be screened in order to find a clone. The identification of a specific clone from a
DNA library can be carried out by exploiting either the sequence of the clone or the structure/
function of its expressed product. The strategy for screening depends upon information about the
gene of interest, the availability of probe and the cloning method used. One of the key elements
required to identify a gene during screening is a probe. A probe is a piece of DNA or RNA that
contains a portion of the sequence complementary to the desired gene for which we are searching.
It is used to detect specific nucleic acid sequences by hybridization (based on complementarity).
The probe can be labelled radioactively (with P32) or non radioactively (biotin, digoxigenin and
fluorescent dyes etc). Probes can be chemically synthesized based on the amino acid sequence of
the protein coded by the gene. Probes can be homologous or heterologous.
Homologous probe - a probe that is exactly complementary to the nucleic acid sequence for
which we are searching; e. g. a human cDNA used for searching a human genomic library.
Heterologous probe - a probe that is similar to, but not exactly complementary to the nucleic acid
sequence for which we are searching; e.g., a mouse cDNA probe used to search a human genomic
library.
There are several methods for screening DNA libraries. Some of the commonly used methods are
described below:
1. Methods based on nucleic acid hybridization
2. Immunochemical methods
3. Screening DNA libraries using PCR
Methods for screening based on detecting a DNA sequence
Screening by hybridization
• Nucleic acid hybridization is the most commonly used method of library screening first
developed by Grunstein and Hogness in1975 to detect DNA sequences in transformed colonies
using radioactive RNA probes.
• It relies on the fact that a single-stranded DNA molecule, used as a probe can hybridize to its
complementary sequence and identify the specific sequences.
• This method is quick, can handle a very large number of clones and used in the identification of
cDNA clones which are not full-length (and therefore cannot be expressed).
The commonly used methods of hybridization are,
a) Colony hybridization
b) Plaque hybridization.

Colony Hybridization
Probes used for hybridization
DNA or synthetic oligonucleotide probes can be used for identification of a clone from a genomic
library. A common method of labeling probes is the incorporation of a radioactive or other marker
into the molecule. A number of alternative labeling methods are also available that involve an
amplification process to detect the presence of small quantities of bound probe and avoid the use
of radioactivity. These methods involve the incorporation of chemical labels such as digoxigenin
or biotin into the probe which can be used for detection
Colony hybridization Colony hybridization, also known as replica plating, allows the screening of
colonies plated at high density using radioactive DNA probes. This method can be used to screen
plasmid or cosmid based.

Plaque hybridization
Plaque hybridization, also known as Plaque lift, was developed by Benton and Davis in
1977 and employs a filter lift method applied to phage plaques. This procedure is successfully
applied to the isolation of recombinant phage by nucleic acid hybridization and probably is the
most widely applied method of library screening. The method of screening library by plaque
hybridization is described below-
• The nitrocellulose filter is applied to the upper surface of agar plates, making a direct contact
between plaques and filter.
• The plaques contain phage particles, as well as a considerable amount of unpackaged
recombinant DNA which bind to the filter.
• The DNA is denatured, fixed to the filter, hybridized with radioactive probes and assayed by
autoradiography.
Advantages
• This method results in a ‘cleaner’ background and distinct signal (less background probe
hybridization) for λ plaque screening due to less DNA transfer from the bacterial host to the
nitrocellulose membrane while lifting plaques rather than bacterial colonies.
• Multiple screens can be performed from the same plate as plaques can be lifted several times.
• Screening can be performed at very high density by screening small plaques. High-density
screening has the advantage that a large number of recombinant Genetic Engineering &
Applications clones can be screened for the presence of sequences homologous to the probe in a
single experiment.
.
Immunological screening
This involves the use of antibodies that specifically recognize antigenic determinants on
the polypeptide. It does not rely upon any particular function of the expressed foreign protein, but
requires an antibody specific to the protein. The secondary antibody recognizes the constant
region of the primary antibody and is, additionally, conjugated to an easily assayable enzyme (e.g.
horseradish peroxidase or alkaline phosphatase) which can be assayed using colorimetric change
or emission of light using X-ray film.
• In this technique, the cells are grown as colonies on master plates and transferred to a solid
matrix. • These colonies are subjected to lysis releasing the proteins which bind to the matrix.
• These proteins are treated with a primary antibody which specifically binds to the protein (acts as
antigen), encoded by the target DNA. The unbound antibodies are removed by washing.
• A secondary antibody is added which specifically binds to the primary antibody removing the
unbound antibodies by washing.
• The secondary antibody carries an enzyme label (e.g., horse radishperoxidase or alkaline
phosphatase) bound to it which converts colorless substrate to colored product. The colonies with
positive results (i.e. colored spots) are identified and subcultured from the master plate.
Screening by PCR
PCR screening is employed for the identification of rare DNA sequences in complex
mixtures of molecular clones by increasing the abundance of a particular sequence. It is
possible to identify any clone by PCR only if there is available information about its
sequence to design suitable primers.
Preparation of a library for screening by PCR can be done by following ways-
● The library can be plated as plaques or colonies on agar plates and individually inoculated
into the wells of the multi-well plate. However it is a labor intensive process and can lead to
bias in favor of larger colonies or plaques.
● The alternative method involves diluting the library. It involves plating out a small part of
the original library (the packaging mix for a phage library, transformation for a plasmid
library) and calculating the titer of the library. A larger sample is diluted to give a titer of 100
colonies per mL. Dispensing 100 μL into each well theoretically gives 10 clones in each well.
These are then pooled and PCR reactions are carried out with gene-specific primers flanking
a unique sequence in the target to identify the wells containing the clone of interest. This
method is often used for screening commercially available libraries.

BAC (BACTERIAL ARTIFICAL CHROMOSOME)

• Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone
very large DNA fragments ranging in size from 100 to 300 kb.
• BACs basically have marker like sights such as antibiotic resistance genes and a very stable
origin of replication (ori) that promotes the distribution of plasmid after bacterial cell division
and maintaining the plasmid copy number to one or two per cell.
• BACs are basically used in sequencing the genome of organisms in genome projects
(example: BACs were used in human genome project).
• Several hundred thousand base pair DNA fragments can be cloned using BACs.
 Bacterial Artificial Chromosome or BAC is circular DNA plasmids constructed with
the origin of replication of E.coli. F' exists as an extra-chromosomal plasmid.
BAC is developed as the first large insert cloning system to provide the construction of DNA
libraries to analyze genomic data. Bacterial artificial chromosomes were developed to study
the genetics and functionality of viruses like the Herpes virus. Since BAC technology has
been promoted in every sector of genetics such as in fingerprinting, human genome
sequencing, development of vaccines, and transgenic techniques.
Bacterial Artificial Chromosome is a DNA construct based on fertility plasmid (F
plasmid) that is used for transformation and cloning in bacteria, primarily E.coli. F plasmids
contain partition genes that ensure the even distribution of plasmids after cell division. The
BAC typically inserts a size of about 150-350 kbp.
BAC is usually utilized to sequence the genome of different organisms for example
Human genome project. A small segment of DNA of an organism is applied as an insert in
BAC and then sequenced. Then the sequenced DNA gets arranged on silicon leading to the
complete genomic sequence of the organism.

Bacterial Artificial chromosome Components

BAC contains basic components in their constructs.

1. For replication of plasmid a regulation of copy number repE is present in the BAC
construct
2. parA and par B are used for partitioning fertility plasmid DNA to daughter cells
during bacterial cell division. It also provides stability and maintenance d BACs.
3. A selectable marker for resistance to antibiotics. Some BAC strains also
contain lacZ at their cloning site for selection between blue or white colonies.
4. T7and Sp6 are used as phage promoters that help in the transcription of inserted
genes. They have also flanked by GC-rich restriction enzyme sites for excision.
How to make BAC libraries
For the making of the BAC library, first, you have to isolate cells that contain the
DNA you want to store. Then mix these cells with hot agarose solution.
Then pour the liquid mixture into a mold to produce a set of a small box, each box with
thousands of isolated cells. Then these cells were treated with specific enzymes to
disintegrate their cell walls and release DNA into the agarose gel
Then the add restriction enzyme cuts the DNA into small fragments of 200,000 base
pairs in length. Then DNA is inserted into a comb containing holes in a slab of agarose gel.
Then these fragments of DNA are separated based on their size by the electrophoresis
process.
On one slab of agarose gel, a solution of DNA ladder or markers is inserted.
Then, the DNA fragments are cut out and extracted out of the gel. These extracted
DNA fragments are then inserted into Bacterial artificial chromosome vectors using a ligase
enzyme that joins the two segments of DNA together. Now, we can call them BAC clones.
These BAC clones are then inserted into the host cell usually E.coli bacterium. The
bacteria are then distributed on nutrient-rich plates that allow only the bacteria carrying BAC
clones to grow on the medium.

Applications
o BAC vectors have been used in studying large double-stranded DNA viruses for
academic research and d as a tool to develop advanced vaccines.
o BAC are utilized for the construction of genomic libraries. They have been used for
basic science research to industrial research like animal husbandry.
o BAC are also used in determining phylogenetic lineage between different species.
o They are also used in the study of horizontal gene transfer.

Advantages of BACs:
• They are capable of accommodating large sequences without any risk of rearrangement.
• BACs are frequently used for studies of genetic or infectious disorders.
• High yield of DNA clones is obtained.

Disadvantages of BACs:
• They are present in low copy number.
• The eukaryotic DNA inserts with repetitive sequences are structurally unstable in BACs
often resulting in deletion or rearrangement.

YEAST ARTIFICIAL CHROMOSOMES (YACS)

 Yeast artificial chromosomes (YACs) are genetically engineered chromosomes
derived from the DNA of the yeast.
 It is a human-engineered DNA molecule used to clone DNA sequences in yeast cells.
 They are the products of a recombinant DNA cloning methodology to isolate and
propagate very large segments of DNA in a yeast host.
 By inserting large fragments of DNA, the inserted sequences can be cloned and
physically mapped using a process called chromosome walking.
 The amount of DNA that can be cloned into a YAC is, on average, from 100 to
2000kb.
  However, as much as 1 Mb (mega, 106) can be cloned into a YAC.

The primary components of a YAC are the ARS, centromere, and telomeres
from Saccharomyces cerevisiae.
ARS: This sequence in YAC is an autonomously replicating sequence.
CEN: This represents the yeast centromere region. This ensures chromosome partitioning
between two daughter cells and a selective marker gene.
TEL: TEL region provides the telomeres. They are not complete telomere sequences but
once they go inside the yeast nucleus they act as seeding sequences in which telomeres build.
Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are
utilized to select transformed yeast cells. Without these sequences, the chromosome will not
be stable during extracellular replication, and would not be distinguishable from colonies
without the vector.
Structure of Yeast Artificial Chromosomes
A yeast artificial chromosome cloning vector consists of two copies of a yeast telomeric
sequence (telomeres are the sequences at the ends of chromosomes), a yeast centromere, a
yeast ars (an autonomously replicating sequence where DNA replication begins), and
appropriate selectable markers.
Working Principle of Yeast Artificial Chromosomes
The yeast artificial chromosome, which is often shortened to YAC, is an artificially
constructed system that can undergo replication. The design of a YAC allows extremely large
segments of genetic material to be inserted. Subsequent rounds of replication produce many
copies of the inserted sequence, in a genetic procedure known as cloning.
 The principle is similar to that for plasmids or cosmids.
 The experimenter introduces some typical elements that are necessary for correct
replication.
 In the case of YACs, the replication origins are the centromeres and telomeres of the
yeast chromosomes, which must be inserted into the DNA being cloned.
 The constructs can be transformed in yeast Spheroplast and are then replicated there.
 In contrast to the vectors, YACs are not circular; they are made of linear DNA.
Process of Yeast Artificial Chromosomes
  YAC vector is initially propagated as circular plasmid inside bacterial host utilizing
bacterial ori sequence.
 The circular plasmid is cut at a specific site using restriction enzymes to generate a
linear chromosome with two telomere sites at terminals.
 The linear chromosome is again digested at a specific site with two arms with
different selection marker.
 The genomic insert is then ligated into YAC vector using DNA ligase enzyme.
 The recombinant vectors are transformed into yeast cells and screened for the
selection markers to obtain recombinant colonies.

Advantages of Yeast Artificial Chromosomes

 Yeast artificial chromosomes (YACs) provide the largest insert capacity of any
cloning system.
  Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs
(yeast episomal plasmids), have advantageous over bacterial artificial chromosomes
(BACs). They can be used to express eukaryotic proteins that require post-
translational modification.
 A major advantage of cloning in yeast, a eukaryote, is that many sequences that are
unstable, underrepresented, or absent when cloned into prokaryotic systems, remain
stable and intact in YAC clones.
 It is possible to reintroduce YACs intact into mammalian cells where the introduced
mammalian genes are expressed and used to study the functions of genes in the
context of flanking sequences.
Uses of Yeast Artificial Chromosomes
 Yeast artificial chromosomes (YACs) were originally constructed in order to study
chromosome behavior in mitosis and meiosis without the complications of
manipulating and destabilizing native chromosomes.
 YACs representing contiguous stretches of genomic DNA (YAC contigs) have
provided a physical map framework for the human, mouse, and even Arabidopsis
genomes.
 YACs are extremely popular for those trying to analyze entire genomes.
Limitations of Yeast Artificial Chromosomes
 A problem encountered in constructing and using YAC libraries is that they typically
contain clones that are chimeric, i.e., contain DNA in a single clone from different
locations in the genome.
 YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of
the cloned DNA. As a result, each YAC clone must be carefully analyzed to be sure
that no rearrangements of the DNA have occurred.
 The efficiency of cloning is low (about 1000 clones are obtained per microgram of
vector and insert DNA).
 YACs have been found to be less stable than BACs.

CHROMOSOME WALKING
When a probe is used for the identification of a gene sequence in a genomic library,
the probe may hybridize with a number of clones, each carrying a part of a large gene
fragmented during preparation of genomic library. If we obtain partial digests (by digesting
the DNA only partially) from the genome, different genomes (from large number of cells)
may give fragments which have overlapping sequences, because sites cleaved in different
genomes of the same organism, will differ being random. Since none of these fragments may
have its entire sequence represented in the probe, overlapping sequences may be used to
construct the original genomic sequence. Identification of fragments with an overlapping
sequence may be a key to the reconstruction or characterization of large chromosome regions.
This is achieved by the technique popularly called chromosome walking.
Introduction
Chromosome walking is a method of positional cloning used to find, isolate, and
clone a particular allele in a gene library. Chromosome Walking was developed by Welcome
Bender, Pierre Spierer, and David S. Hogness in the early 1980's. There are nearly half a
dozen positional cloning tests that are done prior to a chromosome walk. Each clone in the
cosmic library has a DNA insert of 50 KB.The walking starts at the closest gene that has
already been identified, known as a marker gene.
Once the markers on either side of an unmapped sequence are found, the chromosome
walk can begin from one of the markers. Each successive gene in the sequence is tested
repeatedly, known as overlap restrictions and mapped for their precise location in the
sequence. Eventually, walking through the genes reaches the mutant gene in an unmapped
sequence that binds to a fragment of a gene of that particular disease. The testing on each
successive clone is complex, time-consuming, and varied by species. This series of
overlapping clones could for example consist of Bacterial Artificial Chromosomes.
Hybridization Probe
A more straightforward approach thus is to use the insert DNA from the starting clone as a
hybridization probe to screen all the other clones in the library. Positive hybridization signals
that are given by clones, whose inserts overlap with the probe, are used as new probes to
continue the walk. There are about 96 clones that a library consists of and each clone contains
a different insert. A probe may have a genome wide repetition of sequences. This can be
reduced by blocking the repeat sequence with pre-hybridization with unlabeled genomic
DNA. But this isn’t that affective solution especially in the case when high capacity vectors
such as BACs or YACs are used in the walk. Therefore for chromosome walks with human
DNA which have a high rate of repetition, intact inserts are not used in general. Instead the
probe is taken from the end of an insert which has a lesser chance of repetition. The walk can
also be sped up by using the PCR instead of hybridization.

Fig A. The technique of walking through successive hybridization between chromosome

overlapping genomic clones.
 Fig B. Cloning strategy of chromosome walking process
The technique of chromosome walking involves the following steps : (i) from the
genomic library select a clone of interest (identified by a probe) and subclone a small
fragment from one end of the clone (there is a technique available to subclone a fragment
from the end); (ii) the subcloned fragment of the selected clone may be hybridized with other
clones in the library and a second clone hybridizing with the subclone of the first clone is
identified due to presence of overlapping region; (iii) the end of the second clone is then
subcloned and used for hybridization with other clones to identify a third clone having
overlapping region with the subcloned end of the second clone; (iv) third clone identified as
above is also subcloned and hybridized with clones in the same manner and the procedure
may be continued; (v) restriction map of each selected clo.ne may be prepared and compared
to know the regions of overlapping as shown in Figure 40.11, so that identification of new
overlapping restriction sites will amount to walking along the chromosome or along a long
chromosome segment.
Regions of chromosome approaching 1000kb have been mapped following the above
technique. Restriction maps of entire chromosomes can be prepared in this manner following
the technique of chromosome walking.
Application
 This technique can be used for the analysis of genetically transmitted diseases, to look
for mutations.
 Chromosome Walking is used in the discovery of single-nucleotide polymorphism of
different organisms.

Disadvantages
 There is a limitation to the speed of chromosome walking because of the small size of
the fragments that are to be cloned.
 Another limitation is the difficulty of walking through the repeated sequence that are
scattered through the gene.
 If the markers were too far away, it simply was not a viable option.
 Additionally, chromosome walking could easily be stopped by unclonable sections of
DNA.
 A solution to this problem was achieved with the advent of chromosome jumping
(Marx, 1989), which allows the skipping of unclonable sections of DNA.