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Papers reporting the results of genome mapping and sequencing


projects now appear in the scientific literature at the rate of every
fortnight. Cloning is an essential part of many projects. Infact much of
the attention is given on mapping and sequencing the genomes of various
organisms. The approach is simple, to understand the proper functioning
and cast of the genome so that humans can eliminate many disorders and
diseases.

There are several reasons why single gene cloning is still an


important part of molecular biology experiments. One such important
reason is that there remain many genomes that yet to be mapped or
sequenced. The other side is that, the genome sequences reveal only part
of the information available for a given gene. In contrast, cDNA
sequences, which are reverse transcribed from mRNA, reveal expression
profiles in different cell types, developmental stages and in response to
natural or experimentally stimulated external stimuli. Moreover, for
higher organisms cDNA sequences provide useful information about
splice isoforms and their abundance in different tissues and
developmental stages. A further reason is that many cloning strategies
reveal extra functionally annotating genomes always lags way behind the
structural annotation phase, and gene-cloning strategies therefore remain
of value for the elucidation of gene function.

A genomic library is a collection of bacteria which have been


genetically engineered to hold the entire DNA of an organism. The size

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of the library varies, depending on how the DNA is stored in the bacteria,
and the length of the genome of the organism. Genomic libraries are used
in genetic research all over the world in various lab facilities. Companies
which manufacture genomic libraries can provide them by special order
to researchers. With complete information about this for a specific
organism, researchers can perform a variety of experiments on the DNA
to determine the actions and interactions of separate genes along the
strand. They can also compare the genomic library of healthy and
unhealthy individuals of the same species to see where differences in
genetic coding may have led to maladaptive mutations.

In physical reality, a genomic library for humans is a collection of


bacteria, typically E. coli, each carrying a manageable and usable snippet
of DNA from the human genome. The DNA is prepared by digesting it
with a restriction enzyme, then repackaging the separated segments of the
DNA for insertion into the bacteria using lambda phage vectors. This
creates a basic unamplified library. An amplified library is one where the
bacteria have been allowed to multiply and create additional copies of
each section of the DNA.

A cDNA library is a combination of cloned cDNA (complementary


DNA) fragments inserted into a collection of host cells, which together
constitute some portion of the transcriptome of the organism. cDNA is
produced from fully transcribed mRNA found in the nucleus and
therefore contains only the expressed genes of an organism. Similarly,
tissue specific cDNA libraries can be produced. In eukaryotic cells the
mature mRNA is already spliced, hence the cDNA produced lacks introns
and can be readily expressed in a bacterial cell. While information in
cDNA libraries is a powerful and useful tool since gene products are
easily identified, the libraries lack information about enhancers, introns,
and other regulatory elements found in a genomic DNA library.

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A genomic library is a collection of plasmid clones or phage


lysates containing recombinant DNA molecules so that the sum of total
of DNA inserts in this collection, ideally, represents the entire genome of
the concerned organism.

A genomic library contains all the sequences present in the


genome of an organism. The larger the insert of genomic DNA in each
recombinant, the lower the number of recombinants needed to represent
the organism¶s genome completely. For most purposes it is best to use
vectors that will accept large inserts. This effectively means lambda
replacement vectors. Such as EMBL4 or cosmid vectors such as pJB8
and c2R8. Yeast artificial chromosomes, are increasingly widely used as
they can accept inserts even larger than those accepted by cosmids. For
small genomes, lambda insertional vectors or plasmids may be suitable.

Construction of a genomic library:

1)‘The key in generating a high quality library usually lies in the


preparation of the insert DNA. The first step is the isolation of
genomic DNA. The procedures vary widely according to the
organism under study. Care is taken that no physical damage to the
DNA is done, so that it is of high molecular weight and as free of
nicks as possible. If the aim is to prepare a nuclear DNA library,
total DNA is often used, leaving the DNA whatever is present in the
mitochondria or chloroplasts, as there is much more nuclear than
organellar material. If the aim is to form an organelle genomic
library, it would be wise to purify the organelles away from the
nuclei first and then prepare DNA from them.
2)‘The DNA is then fragmented to a size suitable for ligation into the
vector 20-25 kb for EMBL4. Fragments can be made by using
complete digestion by endonuleases, but a large number of
sequences would not be represented intact in a library. Hence,

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partial digestion is better to use, which could frequently cut the
DNA to generate a random collection of fragments with as suitable
size distribution. The partial digestion can be done by two ways;
one is by decreasing the time for digestion or secondly by
decreasing the concentration of the enzyme. Once the fragments are
prepared, they are subjected to phosphatase enzyme to remove
terminal phosphate groups. This ensures that separate pieces of
insert DNA cannot be ligated together before they are ligated into
the vector. Ligation of separate fragments is undesirable as it would
generate clones containing non-contiguous DNA.
3)‘The vector is prepared.
Different vectors can be used as per the requirement.

Plasmids:

Plasmids used in genetic engineering are called vectors. Plasmids


serve as important tools in genetics and biotechnology labs, where they
are commonly used to multiply or express particular genes. Many
plasmids are commercially available for such uses. The gene to be
replicated is inserted into copies of a plasmid containing genes that make
cells resistant to particular antibiotics and a multiple cloning site, which
is a short region containing several commonly used restriction sites
allowing the easy insertion of DNA fragments at this location. Next, the
plasmids are inserted into bacteria by a process called transformation.
Then, the bacteria are exposed to the particular antibiotics. Only bacteria
which take up copies of the plasmid survive, since the plasmid makes
them resistant. In particular, the protecting genes are expressed and the
expressed protein breaks down the antibiotics. In this way the antibiotics
act as a filter to select only the modified bacteria. Now these bacteria can
be grown in large amounts, harvested and lysed to isolate the plasmid of
interest.

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Fig: An plasmid vector (pUC18).

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Lambda (Ȝ) Phage Vectors ±

The Ȝ genome contains an origin of replication, genes for head


and tail proteins and enzymes for DNA replication, lysis and lysogeny,
and single-stranded protruding cohesive ends of 12 bases (5'
GGGCGGCGACCT; the other end is complementary to it, i.e.,
CCCGCCGCTGGA 5').The genome remains linear in the phage head,
but within E. coli cells the two cohesive ends anneal to form a circular
molecule necessary for replication. The sealed cohesive ends are called
cos sites, which are the sites of cleavage during and are necessary for
packaging of the mature DNA into phage heads.
The DNA must be larger than 38 kb and smaller than 52 kb to be
packaged into phage particles. The genes for lysogeny are located in the
segment between 20 and 38 kb; the whole or a part of this segment is
deleted to create Ȝ vectors to
1)‘Accommodate larger DNA inserts and
2)‘ To ensure that the recombinant phage is always lytic.
Several vectors were produced from wild type Ȝ genome by
mutation and recombination in vivo as well as by recombinant
DNA techniques. These vectors have the following two basic
features.
3)‘The vector itself can be propagated as phage in E. coli cells
enabling preparation of vector DNA.
4)‘They contain restriction sites, which allow the removal of the
lysogenic segment and also provide insertion site for the DNA
fragment.
5)‘During annealing and ligation of the DNA insert with the Ȝ vector,
two or more recombinant DNAs may join end-to-end producing a
concatemer, which is the proper precursor for packaging of Ȝ
genome into phage heads.

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Fig: An Phage vector

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   - Cosmids are essentially plasmids that contain a
minimum of 250 bp of Ȝ. DNA which includes

1) The cos site (the sequence yielding cohesive ends) and

2) Sequences needed for binding of and cleavage by terminase so that


under appropriate conditions they are packaged in vitro into empty Ȝ
phage particles.

A typical cosmid has


1) replication origin,
2) unique restriction sites and

3) selectable markers from the plasmid; therefore, selection strategy for


obtaining the recombinant DNA is based on that for the contributing
plasmid.

Cosmid vectors are constructed using recombinant DNA techniques.

The cosmid vectors are opened by the appropriate restriction enzyme at a


unique site, are then mixed with DNA inserts prepared by using the same
enzyme and annealed. Among the several types of products, long
cancatemers are present, which are the appropriate precursors for
packaging in Ȝ. particles.
This procedure selects for long DNA inserts since for packaging the
distance between two cos sites must be between 38 and 52 kb. Cosmids
can accommodate upto 45 kb long DNA inserts. Packaged cosmids infect
host cells like Ȝ particles, but once inside the host they replicate and
propagate like plasmids.

The typical features of cosmids are as follows:


1) they can be used to clone, DNA inserts of upto 45 kb.

2) They can be packaged into A. particles that infect host cells, which is

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many-fold more efficient than plasmid transformation.

3) Selection for recombinant DNA is based on the procedure applicable


to the plasmid making up the cosmid.

4) Finally, these vectors are amplified and maintained in the same


manner as the contributing plasmid.

Fig: An Cosmid vector.

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Bacterial artificial chromosomes:

a bacterial artificial chromosome (BAC) is a DNA construct, based on a


functional fertility plasmid, used for transforming and cloning in bacteria,
usually   F-plasmids play a crucial role because they contain
partition genes that promote the even distribution of plasmids after
bacterial cell division. The bacterial artificial chromosome's usual insert
size is 150-350 kbp, but can be greater than 700 kbp.

A bacterial cloning system based on E. coli F factor was designed which


was capable of cloning fragments of upto 300-350kb. These were
described as bacterial artificial chromosomes (BACs) and are 'user
friendly' being a bacterial system. BAC vectors are superior to other
bacterial systems, based on high to medium copy number of replicons,
since they show structural instability of inserts, deleting or rearranging
portions of cloned DNA.
However, the F factor has regulatory genes that regulate its own
replication and controls its copy number. These regulatory genes include
(I) oriS and repE which mediate unidirectional replication and (ii) parA
and parR, which maintain the copy number to 1 or 2 per E. coli genome.
These essential genes of F factor are incorporated in every BAC vector
(pBAC), which also has a chloroamphenicol resistance gene as a marker
and a cloning segment.

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Yeast artificial chromosomes (YAC¶S):

A yeast artificial chromosome (YAC) is a vector used to clone DNA


fragments larger than 100 kb and up to 3000 kb. YACs are useful for the
physical mapping of complex genomes and for the cloning of large genes.
First described in 1983 by Murray and Szostak, a YAC is an artificially
constructed chromosome and contains the telomeric, centromeric, and
replication origin sequences needed for replication and preservation in
yeast cells. A YAC is built using an initial circular plasmid, which is
typically broken into two linear molecules using restriction enzymes;
DNA ligase is then used to ligate a sequence or gene of interest between
the two linear molecules, forming a single large linear piece of DNA.

Yeast expression vectors, such as YACs, YIps (yeast integrating


plasmids), and YEps (yeast episomal plasmids), have an advantage over
bacterial artificial chromosomes (BACs) in that they can be used to
express eukaryotic proteins that require posttranslational modification.
However, YACs have been found to be less stable than BACs, producing
chimeric effects.

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Fig: Yeast artificial chromosomes.


4) The vector and insert are mixed together, ligated, packaged.

5) If necessary the library is amplified. Llibraries using lambda as cloning


vector are usually kept as stock of packaged phage. Samples of this are
later plated out on an appropriate host when needed. Libraries
constructed in plasmid vectors are kept as collections of plasmid
containing cells or as naked DNA that can be transformed into host cells
when needed. With storage, naked DNA may be degraded. Larger
molecules are more likely to be degraded than smaller ones, so larger
recombinants will be selectively lost and average insert size will fail.

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Generally, genomic libraries are screened following their


construction and introduction of the recombinant DNA into and the
desired recombinant clones are selected and used. An amplified genomic
library consists of the recombinant phage lysates or bacterial clones of a
genomic library. The recombinant DNA produced during genomic library
construction is used for transfection or transformation and multiplied in
the host to yield plaques or clones, which are then stored as amplified
genomic library. Each recombinant DNA is amplified, the amplification
is such that the samples of an amplified library can be plated and
screened with different probes on hundreds of occasions. Amplified
library of recombinant phages can later be stored for many days. On the
other hand, bacterial clones containing recombinant clones are relatively
difficult to store and tend to lose viability that is often unacceptable. But
amplification may distort the library since DNA insert size and sequence
may affect replication of phage, cosmid or plasmid. As a result, particular
DNA inserts may increase or decrease in frequency, and may even be lost
from the library.


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When a library represents only a part of the genome, it is called a


subgenomic library. For example, single chromosomes isolated by flow
cytometry have been used to prepare chromosome-specific libraries.
Particular regions of chromosomes have been microdissected and used
for cloning, for example a specific region of salivary gland chromosome
of 3 
 and specific bands of chromosomes. Such libraries provide
chromosome specific or even chromosome region-specific sequences.
But they require much labour work and are even difficult to construct,
and are often prone to contamination with inappropriate DNA.

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1. Genomic library construction is the first step in any DNA sequencing


projects.
2. Genomic library helps in identification of the novel pharmaceutically
important genes.
3. Genomic library helps in identification of new genes which were silent
in the host.
4. It helps us in understanding the complexity of genomes.

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A cDNA library is a population of bacterial transformants or phage


lysates in which each mRNA isolated from an organism or tissue is
represented as its cDNA insertion in a plasmid or a phage vector. A
cDNA library is a combination of cloned cDNA (complementary DNA)
fragments inserted into a collection of host cells, which together
constitute some portion of the transcriptome of the organism. cDNA is
produced from fully transcribed mRNA found in the nucleus and
therefore contains only the expressed genes of an organism. Similarly,
tissue specific cDNA libraries can be produced. In eukaryotic cells the
mature mRNA is already spliced, hence the cDNA produced lacks introns
and can be readily expressed in a bacterial cell. While information in
cDNA libraries is a powerful and useful tool since gene products are
easily identified, the libraries lack information about enhancers, introns,
and other regulatory elements found in a genomic DNA library.

Isolation of mRNA:

For the isolation of mRNA, total RNA is first extracted from a suitable
organism or tissue. The amount of desired mRNA in this sample is then
increased by using one of the following procedures-

1) Chromatography on poly-U sepharose or oligo-T cellulose, which


retains mRNA molecules since they have 3¶ poly tails.

2) In some specific cases, density gradient centrifugation can be used to


increase the frequency of desired mRNA molecules.

3) Some genes are expressed only in specific tissues, e.g. seed storage
protein genes in developing seeds, chicken ovalbumin gene in oviduct,
globin gene in erythrocytes, insulin gene in ȕ cells of pancreas etc.
Therefore, mRNA preparations from such tissues are exceptionally rich
in the concerned mRNA.


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The RNaseH method:

In this method a complementary DNA strand is synthesized using


reverse transcriptase to make an RNA:DNA duplex, and the RNA strand
is then nicked and replaced by DNA. The first step is to anneal a
chemically synthesized oligo-dT primer to the 3¶ poly A tail of the RNA.
The primer is typically 10-15 residues long and primes synthesis of the
first DNA strand with reverse transcriptase and deoxyribonucleiotides.
This leaves an RNA:DNA duplex, and the next step is to replace the
RNA strand with the DNA strand. The difficulty is finding a way to
prime synthesis using the DNA strand as template. Annealing oliogo-dA
to the oligo-dT incorporated during synthesis of the first strand would be
no use; the oligo-dT is at the 5¶ of the DNA template molecule, but
synthesis must start at 3¶ end. The RNase nicks the RNA leaving a free
3¶-hydroxyl groups and DNA that can then be made using these primers.
As DNA chains are synthesized, any molecules that are base paired to the
template further down are displaced by the polymerase. This leaves
DNA:DNA duple, perhaps with a small region of RNA including any 5¶
cap at one end.


Fig: RNaseH method.

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Self priming method:

A self priming method involves a second-strand cDNA synthesis


method that takes advantage of both the very high processivity and the
very high 3ƍ exonuclease activity of T7 DNA polymerase. The first strand
is synthesized with reverse transcriptase using oligo(dT) as a primer.
After alkaline hydrolysis of the mRNA template, a tract of dT residues is
synthesized with terminal transferase at the 3ƍ end of the first strand. The
second strand is synthesized using oligo(dA) as a primer. Several
oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3ƍ
exonuclease activity of T7 DNA polymerase is very high, the region of
the tract annealed to these oligo(dA) molecules is digested. However, the
region of the tract annealed to the very oligo(dA) molecule used as a
primer for second-strand synthesis is protected. The resulting cDNA
molecules could be cloned with a high efficiency.

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Tailing and priming method:

The poly(A) tail at the 3ƍ end of mRNA seems to have protective function
against exoribonuclease degradation and is involved in initiation of
translation. Stability and translatability of mRNA has been directly
correlated with the length of the polyA tail that is added to the primary
transcript in the nucleus. Upon infection of plus-strand RNA viruses,
their genomes function in two ways: initially, the RNA serves as template
for translation yielding RNA replication factors and subsequently for
minus-strand RNA synthesis, which proceeds in the opposite direction.
Recently, it was shown that picornavirus translation is strongly stimulated
by their polyA tail. Moreover, the crucial importance of the poly(A) tail
in the replication of picornaviruses was deduced from the observation
that     RNA transcripts with a short poly(A) tail had a reduced
specific infectivity. In order to further understand the role of the 3ƍ polyA
tail in the regulation of both translation and replication of plus-strand
RNA viruses, it is of crucial interest to follow changes in the tail length
during the course of viral replication.
Recently, various methods based on reverse transcription polymerase
chain reaction (RT±PCR) amplification have been employed to assess the
polyadenylation state of mRNA [PAT, polyA test assay, reviewed in.
Since in these assays oligodT adaptor primers are used which can anneal
at any position within the polyA tail, the products of RT might not
represent the complete polyA tail. Here, we describe a new PCR-based
oligoG-tailing method in which the 3ƍ end of the mRNA is immediately
preserved from degradation by the enzymatic addition of an oligoG tail.
With this step a polyA±oligoG junction is generated which serves as
specific target for the amplification of the 3ƍ end of the viral genome with
the universal reverse primer oligo(dC9T6) and a gene-specific forward
primer. The universal antisense primer also ensures that only RNA
molecules terminating with adenosine residues are amplified. The
subsequent sequencing of the RT±PCR product allowed the accurate
polyA tail length quantification. Using this method, the poly(A) tail
co
length of hepatitis A virus (HAV) RNA rescued after transfection of  
  transcripts with a defined numbers of adenosine residues was
determined by sequencing.

Fig: Tailing and priming method.

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a     a    

There are no introns, so there is no danger of pieces of


your gene being chopped onto separate clones; and the library is
(hopefully) enriched for your gene, since instead of one or two copies, as
in the genomic library, you have as many copies as the cell could produce
mRNA's for that gene. So most molecular biologists, when searching for
a new gene, start by screening a cDNA library from a tissue or organism
that they suspect is actively using that gene.

a     a    

{‘ Discovery of novel genes.


{‘ Cloning of full-length cDNA molecules for     study of gene
function.
{‘ Study of the repertoire of mRNAs expressed in different cells or
tissues.
{‘ Study of alternative splicing in different cells or tissues.
{‘ Determining the complete genome sequence of a given organism.
{‘ Serving as a source of genomic sequence for generation of
transgenic animals through genetic engineering.
{‘ Study of the function of regulatory sequences    
{‘ Study of genetic mutations in cancer tissues.

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Colony or plaque hybridisation:

Once a genomic library or cDNA library is available, we may like


to use it for isolation of a gene sequence. This can be achieved by colony
hybridization technique illustrated. In this technique, bacteria carrying
chimeric vectors are grown into colonies, which are lysed on
nitrocellulose filters.

Their DNA is denatured in situ and fixed on the filter, which is


hybridized with a radioactively labeled probe carrying a sequence related
to the gene to be isolated (usually a cloned cDNA for screening of a
genomic library).
Colonies carrying this sequence will be identified by dark spots after
autoradiography, so that the original chimeric vector carrying the desired
gene sequence can be recovered from one or more colonies in the
original master plate and used for further experiments.

This technique is described as colony hybridization. It is possible that


a probe may identify more than one clones or that a gene is fragmented
in the library. In such a case, one needs to reconstruct the desired
sequence using several overlapping sequences available in the library.
This is a very routine exercise whenever we like to isolate specific
DNA sequences from the genome of a species, or from cDNA derived
from mRNA of a specific tissue of a species. Sometimes the library may
be available not in the form of bacteria transformed with chimeric DNA
molecules, but in the form of chimeric phage particles carrying the
cloned segments.

In such a situation, a bacterial lawn is infected with a mixture of


chimeric phage particles (i.e. the library) and a large number of plaques
develop overnight. These plaques can be treated just like the colonies in
colony hybridization to identify and isolate the chimeric phage particle
carrying the gene of interest. This technique is then described as plaque
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Blue white screening:

The blue-white screen is a molecular technique that allows for the


detection of successful ligations in vector-based gene cloning. DNA of
interest is ligated into a vector. The vector is then transformed into
competent cell (bacteria). The competent cells are grown in the presence
of X-gal. If the ligation was successful, the bacterial colony will be white;
if not, the colony will be blue. This technique allows for the quick and
easy detection of successful ligation, without the need to individually test
each colony. An example of such a vector is the artificially reconstructed
plasmid pUC19.

Molecular Mechanism:

Cloning, alongside PCR, is one of the most common techniques in


molecular biology. Blue white screening makes this procedure less time
and labor intensive by allowing for the screening of successful cloning
reactions through the colour of the bacterial colony.

The molecular mechanism for blue/white screening is based on a


genetic engineering of the   operon in the 
 
  laboratory
strain serving as a host cell combined with a subunit complementation
achieved with the cloning vector. The vector (e.g. pBluescript) encodes
the Į subunit of LacZ protein with an internal multiple cloning site
(MCS), while the chromosome of the host strain encodes the remaining ȍ
subunit to form a functional ȕ-galactosidase enzyme. The MCS can be
cleaved by different restriction enzymes so that the foreign DNA can be
inserted within the  Į gene, thus disrupting the production of
functional ȕ-galactosidase. The chemical required for this screen is X-gal,
a colourless modified galactose sugar that is metabolized by ȕ-
galactosidase to form an insoluble product (5-bromo-4 chloroindole)
which is bright blue, and thus functions as an indicator. Isopropyl ȕ-D-1-
thiogalactopyranoside (IPTG), which functions as the inducer of the Lac
operon, can be used in some strains to enhance the phenotype, although it
is with many common laboratory strains unnecessary. The hydrolysis of
colourless X-gal by the ȕ-galactosidase causes the characteristic blue
colour in the colonies; it shows that the colonies contain vector without

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insert. White colonies indicate insertion of foreign DNA and loss of the
cells' ability to hydrolyse the marker.

Bacterial colonies in general, however, are white, and so a bacterial


colony with no vector at all will also appear white. These are usually
suppressed by the presence of an antibiotic in the growth medium. A
resistance gene on the vector allows successfully transformed bacteria to
survive despite the presence of the antibiotic.

The correct type of vector and competent cells are important


considerations when planning a blue white screen.

It is also important to understand the lac operon is regulated by cAMP


levels and the binding of cAMP to CAP. This CAP-cAMP complex
promotes the binding of RNA polymerase to the lac promoter, which
leads to transcription of the lac genes. cAMP levels are regulated by the
cell's incorporation of glucose. Since most bacteria preferentially utilize
glucose even in the presence of lactose, the lac genes will only be turned
on when glucose levels drop low enough to allow the CAP-cAMP
complex to form.

Disadvantage:

Some white colonies may not contain the recombinant plasmid that the
researcher is looking for since it only takes a small piece of DNA to be
ligated into the vector's Multiple Cloning Site that changes the reading
frame for LacZĮ, thus preventing its expression. Furthermore, some
linearized vector may get transformed into the bacteria, the ends
"repaired" and ligated together such that no LacZĮ is produced. As a
result, these cells cannot convert X-gal to the blue substance. On the
other hand, in some cases, blue colonies may contain the insert. This
occurs when the insert is "in frame" with the LacZĮ gene and it does not
have a STOP codon. This could sometimes lead to the expression of a
fusion protein that is still functional as LacZĮ. Lastly, the correct
recombinant construct may give light blue colonies that are distinguished
from the dark blue non-recombinants and white constructs that were
"repaired" as described above.

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Fig: pUC18 A common cloning vector.

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Fig: Bacterial colonies transformed with pUC18.

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ù‘ The genomic library contains DNA fragments representing the


entire genome of an organism.
ù‘ The cDNA library contains only complementary DNA molecules
synthesized from mRNA molecules in a cell.
ù‘ They are important as they provide a gateway for cloning.
ù‘ Even a single gene of interest can be easily isolated and cloned.
ù‘ It allows us to properly notify the expressions of various genes
present in the genome.
ù‘ Genomic library construction is the first step in any DNA
sequencing projects.
ù‘ Genomic library helps in identification of the novel
pharmaceutically important genes.
ù‘ Genomic library helps in identification of new genes which were
silent in the host.
ù‘ It helps us in understanding the complexity of genomes.
ù‘ Entire genome of an organism is used in preparing of a genomic
library.
ù‘ Genomic libraries are not useful while working with eukaryotes.
ù‘ Screening sometimes becomes difficult.
ù‘ However, genomic libraries allow us to study the genome sequence
of a particular gene.
ù‘ There are no introns, so there is no danger of pieces of your gene
being chopped onto separate clones; and the library is
(hopefully) enriched for your gene, since instead of one or two
copies, as in the genomic library, you have as many copies as the
cell could produce mRNA's for that gene. So most molecular
biologists, when searching for a new gene, start by screening a
cDNA library from a tissue or organism that they suspect is actively
using that gene.

Ôo
Ñ  

ù‘ B.D.Singh, Biotechnology expanding horizons, 2009 edition,


Kalyani publishers, 25-36.
ù‘ Ernst-L.Winnacker, From genes to genomes, 2003 edition, Panima
publishing house, 32-46.
ù‘ Jeremy.W.Dale and Malcolm von Schantz, From genes to genomes
concept and applications, 2002 edition, British library publications,
99-116.
ù‘ Julia Lodge , Pete Lund and Steve Mirchin, Gene cloning principles
and applications, Taylor and francis group, 85-141.

Œ