report on genomic and cdna libraries

8/7/2019 Report on Genomic and cDNA libraries

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Introduction:

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 cDNAlibrary 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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Genomic libraries:

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 ofthe 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 organisms 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 pJB8and 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 thenuclei 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 makecells 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 canbe 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 () Phage Vectors

The 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 isdeleted to create 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 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 vector,

two or more recombinant DNAs may join end-to-end producing a

concatemer, which is the proper precursor for packaging of

genome into phage heads.


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Fig: An Phage vector


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Cosmid Vectors - Cosmids are essentially plasmids that contain a

minimum of 250 bp of . 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

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 . 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 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:

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

functional fertility plasmid, used for transforming and cloning in bacteria,

usually E-Coli F-plasmids play a crucial role because they containpartition 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 markerand a cloning segment.


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Yeast artificial chromosomes (YACS):

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 thephysical 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.


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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. Librariesconstructed 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.

Amplified genomic libraries:

Generally, genomic libraries are screened following their

construction and introduction of the recombinant DNA into E.coli 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 sequencemay 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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Subgenomic libraries:

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

ofDrosophila 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.

Applications of genomic libraries:

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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cDNA libraries:

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. AcDNA 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 thenincreased 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 cells of pancreas etc.

Therefore, mRNA preparations from such tissues are exceptionally rich

in the concerned mRNA.


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Methods ofsynthesising cDNA:

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-dAto 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.


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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 3 exonuclease activity of T7 DNA polymerase. The first strandis 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 3 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 3

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 3 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 directlycorrelated 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 in vitro RNA transcripts with a short poly(A) tail had a reduced

specific infectivity. In order to further understand the role of the 3 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 (RTPCR) amplification have been employed to assess thepolyadenylation 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 3 end of the mRNA is immediately

preserved from degradation by the enzymatic addition of an oligoG tail.

With this step a polyAoligoG junction is generated which serves as

specific target for the amplification of the 3 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 RTPCR product allowed the accurate

polyA tail length quantification. Using this method, the poly(A) tail


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length of hepatitis A virus (HAV) RNA rescued after transfection of in

vitro transcripts with a defined numbers of adenosine residues was

determined by sequencing.

Fig: Tailing and priming method.


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Advantages of cDNA libraries:

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.

Applications of cDNA libraries:

y Discovery of novel genes.

y Cloning of full-length cDNA molecules forin vitro study of gene

function.

y Study of the repertoire of mRNAs expressed in different cells or

tissues.

y Study of alternative splicing in different cells or tissues.

y Determining the complete genome sequence of a given organism.

y Serving as a source of genomic sequence for generation of

transgenic animals through genetic engineering.

y Study of the function of regulatory sequences in vitro.

y Study of genetic mutations in cancer tissues.


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Sreening of libraries:

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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hybri i ti

i : C l y or pl hybri i tion.


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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 intocompetent 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 inmolecular 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 lac operon in the Escherichia coli laboratory

strain serving as a host cell combined with a subunit complementation

achieved with the cloning vector. The vector (e.g. pBluescript) encodes

the subunit of LacZ protein with an internal multiple cloning site(MCS), while the chromosome of the host strain encodes the remaining

subunit to form a functional -galactosidase enzyme. The MCS can be

cleaved by different restriction enzymes so that the foreign DNA can be

inserted within the lacZ gene, thus disrupting the production of

functional -galactosidase. The chemical required for this screen is X-gal,

a colourless modified galactose sugar that is metabolized by -

galactosidase to form an insoluble product (5-bromo-4 chloroindole)

which is bright blue, and thus functions as an indicator. Isopropyl -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 -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, whichleads 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 theresearcher 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 LacZ, thus preventing its expression. Furthermore, some

linearized vector may get transformed into the bacteria, the ends

"repaired" and ligated together such that no LacZ 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 LacZ gene and it does not

have a STOP codon. This could sometimes lead to the expression of afusion protein that is still functional as LacZ. 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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Fi : LacZ a screenable marker.


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Bacterial colonies transformed with pUC18

blue colonies(contain non-recombinant DNAmolecules)

White colonies(contain recombinant DNAmolecules)

Fig: Bacterial colonies transformed with pUC18.


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Conclusion:

The genomic library contains DNA fragments representing the

entire genome of an organism.

The cDNA library contains only complementary DNA moleculessynthesized from mRNA molecules in a cell.

They are important as they provide a gateway for cloning.

Even a single gene of interest can be easily isolated and cloned.

It allows us to properly notify the expressions of various genes

present in the genome.

Genomic library construction is the first step in any DNA

sequencing projects.

Genomic library helps in identification of the novel

pharmaceutically important genes.

Genomic library helps in identification of new genes which were

silent in the host.

It helps us in understanding the complexity of genomes.

Entire genome of an organism is used in preparing of a genomic

library.

Genomic libraries are not useful while working with eukaryotes.

Screening sometimes becomes difficult.

However, genomic libraries allow us to study the genome sequence

of a particular gene.

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 molecularbiologists, 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.


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Bibliography:

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

Kalyani publishers, 25-36.

Ernst-L.Winnacker, From genes to genomes, 2003 edition, Panimapublishing house, 32-46.

Jeremy.W.Dale and Malcolm von Schantz, From genes to genomes

concept and applications, 2002 edition, British library publications,

99-116.

Julia Lodge , Pete Lund and Steve Mirchin, Gene cloning principles

and applications, Taylor and francis group, 85-141.

report on genomic and cdna libraries

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