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Dr. Monica Garg Mudaser Ahmed Lone RP7905 P7905 10907230

Genomic Library A genomic library is a collection of genes or DNA sequences in the entire genome created using molecular cloning. These libraries are constructed using clones of bacteria or yeast that contain vectors into which fragments of partially digested DNA have been inserted. These bacteria and yeast are subsequently grown in culture and when these microorganisms replicate their genome, they also replicate the vector genome contained within them, that is, they replicate DNA fragments that had been inserted in vectors producing clones of the original DNA.

This collection of clones, in theory, contains all sequences found in the original source, including the sequence of interest. This sequence of interest is identified using screening methods that are very complex and capable of finding the original clone among 10 million starting clones. Construction of Genomic Library The idea of amplifying very large mixtures of DNA fragments and then trying to find the single segment of interest was dramatic. Now it is almost common to use this strategy. The process of subdividing genomic DNA into clonable elements and inserting them into host cells is called creating a library. A complete library, by definition, contains all of the genomic DNA of the source organism and is called as genomic library. A genomic library is a set of cloned fragments of genomic DNA.

The process of creating a genomic library includes four steps.

In the first step the high molecular weight genomic DNA is separated and subjected to restriction enzyme digestion by using two compatible restriction enzymes. In the second step fragments are then fractionated or separated by using agarose gel electrophoresis to obtain fragments of required size .These fragments are then subjected to alkaline phosphatase treatment to remove the phosphate. In the third step, the dephosphorylated insert is ligated into vector which could be a plasmid, phage or cosmid, depending upon the interest of the researcher. In the last step, the recombinant vector is introduced into the host by electroporation and amplified in host

Genomic library is a source of genes and DNA sequences. A genomic library is a set of cloned fragments of genomic DNA. In principle, the gDNA, after the isolation, is subjected to Resriction enzyme for digestion to generate inserts. Depending upon the vector type and genome size of the source, the size of insert is selected .For example, if genome is 2.8 x 106 kb and if it is broken down into fragments of size 4 kb then we require 7 x 105 recombinant clones for the work. If

the same genome is broken down into fragments of size 20 kb then we require 1.4 x 105 recombinants. Thus the fragment size will influence the number of clones to be formed, so that there is 99% probability of success when a desired clone is searched. In reality, the number of clones (f) required for 99% probability is not calculated just by dividing the genome size by size of insert. The number of independent recombinants required in the library must be greater than n. Clarke and Carbon gave equation and is applied. This formula relates the probability (P) of any DNA sequence in a random library of N independent recombinants. N = In(I - P)/In(l -l/f) where, N = number of clones required to represent an ideal library P= probability f= genome size/size of insert Large insert vectors have the advantage that libraries containing fewer clones are needed in order to cover an entire genome .But Larger inserts are prone to deletions or rearrangements. Also, the vectors with large inserts are normally maintained at low copy number even when the vector by itself has high copy number. These vectors are purified by sucrose gradient or by gel

electrophoresis.Later these fragments are cloned into vector for amplification. Before ligation to the vector, the digested genomic DNA is treated with alkaline phosphatase to prevent the coligation of two inserts to a single vector molecule. When a genomic library is constructed, the choice of vector should be carefully considered. Normally phages are used for constructing the genomic library when compared to cosmids and plasmids. Phages can be used for repeated amplification, thus once a genomic library is constructed, it can be used for generations or years together. Secondly, phage recombinants give better and cleaner results when desired clones are identified by colony hybridization. An important problem in genomic library is distortion. Not all recombinants in a population will propagate equally well, as the insert size or sequence may affect the replication of a recombinant phage. Hence when a library is put through an

amplification step, particular recombinants may be increased in frequency or decreasing or totally lost. With the technological development in genomic library construction methods, most of the researchers prefer to create a new library for each screening rather than take the risk of using previously amplified ones. Genomic libraries can be constructed using various hosts like plasmids (insert size up to 15 kb), bacteriophage lambdas (insert size up to 20 kb), cosmids (insert size up to 45 kb), YACs and (insert size up to 2,000 kb), and many more. Some important ones are as follows: Constructing Genomic Libraries Using Plasmids: This is the process of cloning human DNA fragments by inserting them between two EcoRI digested sites of a plasmid cloning vector. After the human DNA fragments are inserted into the plasmid, the plasmids in turn are inserted into bacterial cells. In the end, these host bacterial cells contain their own chromosomal DNA together with plasmid DNA. As these bacteria replicate their chromosomes, they also replicate with them the plasmid cloning vectors. Plasmids are very convenient to work with because they contain one origin of replication, one of more selectable markers (such as a gene that confers resistance to antibiotics), and one or more restriction sites that can be cut and used for ligation of foreign DNA molecules.

Constructing Genomic Libraries Using Bacteriophage Vectors:

The human genome is partially digested with a restriction enzyme like Sau3A in a specific way so that some of the sites are cleaved and others are not. By this way, random cleavage of the sites occurs and a collection of overhanging fragments of length suitable for cloning can be obtained, which are then ligated into bacteriophage lambda arms prepared so that the Sau3A ends of human DNA fragments can be ligated into the vector. The recombinant lambda chromosomes are then packaged into the infectious bacteriophage, and then the library, containing 1 million or more fragments of genomic DNA, can be stored for the

future isolation of many genes. A collection of several hundred thousand phages would represent the entire DNA from the human genome. Making a genomic library in bacteriophage lambda vector:

Phage lambda bind to receptors on the surface of E. coli and injects DNA. Infection by lambda is much more efficient than plasmid transformation can get ~ 109 plaques per microgram of DNA, vs ~ 106 colonies per microgram of plasmid DNA As the bacterial cells lyse, a plaque forms, which spreads as more cells become infected and lyse. The plaque contains the infectious viral particles or virions and each one represents an individual lambda clone. The appearance is of a clear circle in a 'cloud' of bacterial cells (the bacterial 'lawn'). If incubated for long periods, these circles will continue to grow and spread until all bacterial cells are wiped out. To pick a single lambda clone, the plaque is 'punched' out from the agar plate and stored in a holding solution. This can then be used to infect more bacterial cells and to replicate more of the recombinant lambda DNA. Constructing Genomic Libraries with Yeast Artificial Chromosomes (YACs): Large fragments of human DNA (over 500 kb) are generated by partial EcoRI digestion of human genomic DNA. Individual vector arms contain telomeres at one end and EcoRI compatible overhangs on at the other end. Individual vectors also carry a different selectable marker, and one arm also contains a centromere and selectable markers at each end and a yeast chromosome at the other end. The YACs are transferred into yeast, and the selectable markers are used to select only those yeasts that contain a properly constructed YAC.

YAC libraries are used for cloning very large DNA fragments (of more than 1 Mb), and are useful for cloning large genes (such as the 250 kb cystic fibrosis gene) and for creating libraries of large overlapping clones, such as for individual chromosomes isolated from organisms (chromosomal libraries). YACs are yeast artificial chromosomes and are hybrids of bacterial plasmid DNA and yeast DNA. The components required for replication/segregation of natural yeast chromosomes have been combined with E. coli plasmid DNA. YACs are grown in the yeast Saccharomomyces cerevesiae and so contain selectable markers which are suitable for the host system. Rather than antibiotic selection,

yeast selectable markers enable growth of the transformant on selective media lacking specific nutrients. (Non-transformants are unable to grow). The yeast strains that are used are auxotrophic - that is, they are unable to make a specific compound. YACs are not used as extensively anymore because of inherent problems. For instance, YAC clones can contain non-contiguous segments of the genome. This means that two or more DNA fragments from separate parts of the genome can be integrated into an individual .A second problem is that YACs are unstable and frequently lose parts of the DNA during propagation.

Construction genomic libraries with cosmid

Cosmid vectors are hybrids of plasmid and bacteriophage lambda DNA (a small ~5 kb plasmid containing the plasmid origin of replication (ori), an antibiotic resistance gene such as ampr and a suitable restriction site for cloning along with the COS sequence from phage DNA). Because of the COS sequence, cosmid recombinants can be packaged into viral particles (allowing high efficiency transformation). Since most of the DNA has been discarded, it can be replaced by the DNA of interest, so long as it doesn't exceed the 50 kb limit for packaging into the viral head. The insert sizes are of the order of 35-45 kb. Since the recombinant DNA does not encode any lambda proteins, cosmids do not form viral particles (or plaques) but rather forms large circular plasmids and the colonies that arise can be selected on antibiotic plates, like other plasmid DNA transformants. Cosmid clones can be manipulated similarly, allowing ease of plasmid isolation .Since many eukaryotic genes are on the order of 30 - 40 kb, the likelihood of obtaining a DNA clone containing the entire gene sequence is increased significantly when using a cosmid library.

BAC libraries are also used for cloning very large DNA fragments and have been particularly useful for sequencing large genomes. BACs are bacterial artificial chromosomes, and are based on a naturally occurring large bacterial plasmid, the F-factor. BAC vectors can accommodate DNA inserts up to 300 kb, still fairly respectable when needing to clone large genes or map and sequence complex genomes. BACs have several advantages over YACs, which means they are used more extensively now. Most of the human genome has been sequenced using BAC ather than YAC clones.

Many different strategies are available for library screening

The various techniques used for screening to any type of library, genomic or cDNA, and can involve either Nucleic acid hybridization or the PCR. In each case, the design of the probe or primers can be used to home in on one specific clone or a group of structurally related .Both genomic and cDNA libraries can be screened by hybridization Nucleic acid hybridization is the most commonly used method of library screening because it is rapid ,it can be applied to very large numbers of clones, and in the case of cDNA libraries, can be used to identify clones that are not full length .Grunstein & Hogness (1975) developed a screening procedure to detect DNA sequences in transformed colonies by hybridization in situ with radioactive RNA probes. Their procedure can rapidly determine which colony among thousands contains the target sequence.A modification of the

method low screening of colonies plated at a very high density. The colonies to be screened are first replica plated onto a nitro cellulose filter disk that has been placed on the surface of an agar plate prior to inoculation A reference set of these colonies on the master plate is retained. The filter bearing the colonies is removed and treated with alkali so that the bacterial colonies are lysed and the DNA they contain is denatured. The filter is then treated with proteinase K to remove protein and leave denatured DNA bound to the nitro cellulose, for which it has a high affinity, in the form of a DNA-print of the colonies. The DNA is fixed

firmly by baking the filter at 80C. The defining , labeled RNA is hybridized to this DNA and the result of this hybridization is monitored by autoradiography . The PCR can be used as an alternative to hybridization for the screening of genomic and cDNA libraries

The PCR is widely used to isolate specific DNA sequences from uncloned genomic DNA or cDNA, but it is also a useful technique for library screening. As a screening method, the PCR has the same versatility as hybridization, and the same limitations. It is possible to identify any clone by the PCR but only if there is sufficient information about its sequence to make suitable primers. To isolate a specific clone, the PCR is carried out with gene-specific primers that flank a unique sequence in the target. A typical strategy for library screening by the PCR is demonstrated by Takumi &Lodish (1994). Each well is screened by the PCR and positive wells are identified. The clones in each positive well are then diluted into a series in a secondary set of plates and screened again. The process is repeated until wells carrying homogeneous clones corresponding to the gene of interest have been identified. There are also several applications where the use of degenerate primers is favorable. A degenerate primer is a mixture of primers, all of similar sequence but with variations at one or more positions. This is analogous to the use of degenerate oligonucleotides as hybridization probes, and the primers are synthesized in the same way. A common circumstance requiring the use of degenerate primers is when the primer sequences have to be deduced from aminoacid sequences As with oligonucleotides probes, the selection of amino acids with low codon degeneracy is desirable. However, a 128-fold degeneracy in each primer can be successful in amplifying a single copy target from the human genome

Immunological screening uses specific antibodies to detect expressed gene products

Immunological screening involves the use of antibodies that specifically recognize antigenic determinants on the polypeptide synthesized by a target clone. This is one of the most versatile expression cloning strategies because it can be applied to any protein for which an antibody is available. Unlike the screening strategies discussed below, there is also no need for that protein to be functional. The molecular target for recognition is generally an epitope, a short sequence of amino acids that folds into a particular three-dimensional conformation on the surface of the protein. Epitopes can fold independently of the rest of the protein and therefore often form even when the polypeptide chain is incomplete, or when expressed as a fusion with another protein. Importantly, many epitopes can form under denaturing conditions when the overall conformation of the protein is abnormal. The first immunological screening techniques were developed in the late 1970s, when expression libraries were generally constructed using plasmid vectorsThis method exploited the facts that antibodies adsorb very strongly to certain types of plastic, such as polyvinyl, and that IgG antibodies. As usual, transformed cells were platedout on Petri dishes and allowed to form colonies. In order to release the antigen from positive clones, the colonies were lysed, e.g. using chloroform vapor or by spraying with an aerosol of virulent phage .A sheet of polyvinyl that had been coated with the appropriate ntibody was the applied to the surface of the plate, allowing antigen antibody complexes to form. The sheet was then removed and exposed to 125Ilabeled IgG specific to a different determinant on the surface of the antigen(i.e. a determinant not involved in the initial bindingof the antigen to the antibodycoated sheet; The sheet was then washed and exposed to X-rayfilm. The clones identified by this procedure could then be isolated from the replica plate. Note that thissandwich technique is applicable only where two antibodies recognizing different determinants of the same protein are available. However, if the proteinis expressed as a fusion, antibodies that bind to each component of the fusion can be used, efficiently selecting for recombinant molecules.While plasmid libraries have been useful for expression screening (Helfman et al. 1983, Helfman use bacteriophage insertion vectors because these have a higher

capacity and the efficiency of in vitro packaging allows large numbers of recombinants to be prepared and screened. Immunological screening with phage cDNA libraries was introduced by Young & Davies (1983) using the expression vector gt11 which generates fusion proteins with -galactosidase under the

control of the lac promoter. In the original technique, screening was carried out using colonies of induced lysogenic bacteria, which required the production of replica plates as above. A simplification ofthe method is possible by directly screening plaques of recombinant phage. In this procedure the library is plated out at moderately high density(up to 5 104 plaques per 9 cm2 plate), with E. coli strain Y1090 as the host. This E. coli strain over produces the lac repressor and ensures that no expression of cloned sequences (which may be deleterious to the host) takes place until the inducer IPTG is presented to the infected cells. Y1090 is also deficient in the lon protease, hence increasing the stabilityof recombinant fusion proteins. Fusion proteins expressed in plaques are absorbed onto a nitrocellulose membrane overlay and this membrane is processed for antibody screening. When a positive signal is identified on the membrane, the positive plaque can be picked from the original agar plate (a replica is not necessary) and the recombinant phage can be isolated. The original detection method using iodinated antibodies has been superseded by more convenient methods using non-isotopic labels, which are also more sensitive and have a lower background of nonspecific signal. Generally, these involve the use of unlabeled primary antibodies directed against the polypeptide of interest, which are in turn recognized by secondary antibodies carrying an enzymatic label. As well as eliminating the need for isotopes, such methods also incorporate an amplification step, since two or more secondary antibodies bind to the primary antibody. Typically, the secondary antibody recognizes the species-specific constant region of the primary antibody, and is conjugated to alkaline phosphatase (Mierendorf et al. 1987), each of which canin turn be detected using a simple colorimetric assay carried out directly on the nitrocellulose filter. Polyclonal antibodies, which recognize many different epitopes, provide a very sensitive probe for immunological screening, although they may also cross react

antibodies and cloned antibody fragments can alsobe used, although the sensitivity of such reagents is reduced because only a single epitope is recognized South western and north western screening are used to detect clones encoding nucleicacid binding proteins However, the screening is carried out not using an antibody, by incubating the membranes with a radio labeled double-stranded DNA oligonucleotide probe, containing the recognition sequence for the target DNA-binding protein. This technique is called southwestern screening because it combines the principles of Southern and western blots. It has been particularly successful inthe isolation of clones expressing cDNA sequences corresponding to certain mammalian transcription factors .A limitation of this techniqueis that, since individual plaques contain only single cDNA clones, transcription factors that function only in the form of heterodimers or as part of a multimeric complex do not recognize the DNA probe, and the corresponding cDNAs cannot be isolated. Clearly the procedure can also be successful only in cases where the transcription factor remains functional when expressed as a fusion polypeptide. It is also clear that the affinity of the polypeptide for the specific DNA sequence must be high, and this has led to the preferential isolation of certain types of transcription factor .More recently, a similar technique has been used to isolate sequence-specific RNA-binding proteins, in this case using a single-stranded RNA probe. By analogy to the above, this is termed north western screening and has been successful in a number of cases. Both southwestern and north western screening are most efficient when the oligonucleotide contains the binding sequence in multimeric form.This may mean that several fusion polypeptides on the filter bind to each probe, hence greatly increasing the average dissociation time. To minimize non-specific binding, a large excess of unlabeled double-stranded DNA (or single-stranded RNA) is mixed with the specific probe. However, it is usually necessary to confirm the specificity of binding in a second round of screening using the specific oligonucleotide probe and one or more alternative probes containing similar sequences that are not expected to be recognized.As well as

DNA and RNA, a whole range of alternativeligands can be used to identify polypeptides that specifically bind certain molecules. Such techniques are not widely used because they generally have a low sensitivity and their success depends on the preservation of the appropriate interacting domain of the protein when exposed on the surface of a nitrocellulose filter . The yeast two-hybrid system and its derivatives now provide versatile assay formats for many specific types of proteinprotein interaction, with the advantage that such interactions are tested in living cells so the proteins involved are more likely to retain their functional interacting domains.

Applications of Genomic Library 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.

References Books Prescibed :  Gene Manpulation By Primose and Twyan and Genomics by T A Brown