Genetic and Physical Maps

In a genetic map markers are arranged in specific order determined by the frequency of crossing over between them the distance between neighbouring genes is represented in terms of centimorgans. In contrast a physical map deplets only the physical locations of markers the order and the location of various markers ids determined by using physical methods like in situ hybridization RFLP etc. Therefore when a known gene is located in a map by means of in situ hybridization it becomes a part of a physical map mere location of a gene or genes does not make th e map genetic map. To qualify as a genetic map, genetic methods must be used for preparing the map such a map must be prepared by using recombination data. Hence, when molecular markers e.g. RFLP, microsatelites probes etc, are pklace a map using recombination data a genetic map is generated. The physical and genetic maps can be integrated by studying recombination frequencies between the linked marker in appropriate segregating population e.g backcrosses etc. or in random sample inbreds derived from appropriate heterozygotes for the markers in question in case of human beings, this is done by studying the inhertance patterns of the selected markers in group of families choosen for the purpose. Initially, the human genome project selected a group of 59 families for the preparation of primary genetic linkage maps. These families represented from 1,212 meioses. In order to establish a permanent source of DNA, cel lines were established from the individuals of this group of selected families at the Centre for study of Human polymorphism, paris

Genome Maps

A genome map may be defined as a detailed schematic description of the structural and functional organisation of all the chromosomes in the genome of an organism. At present we have mainly two types of maps (1) genetic or linkage maps and (2) physical maps. A genetic map is prepared on the basis of recombination data between carefully selected genetic markers usually ordered into suitable crosses but in case of humans linkage maps have to the prepared using family pedigree data in addition genes could be assigned to a specific chromosome arms using somatic cell hybridization Linkage groups have been assigned to specific chromosomes in the genome by using appropriate cytogenic techniques in conjunction with linkage studies. The chief problem of linkage mapping is the non availibility of a sufficient number of genetic markers to cover the entire genome.
Therefore, search for more abundant markers continued and resulted in various molecular markers, e.g. RFLP, RAPD and STR used in mapping of chromosomes. Chromosome maps depieting the location of these molecular markers, whose genetic function is ordinarily not known, are called physical maps.
In several species genetic maps have been being integrated with the physical maps to yield a highly useful genomic map using which genes of interest can be detected and or isolated with the help of more convenient and highly reliable molecular markers such studies have revealed that in human I centimorgan the distance which allow 1% recombination between two markers represents about 1000 kb of DNA while in mice it is equivalent to about 1600 kb DNA.

Animal Biotechnology

Introduction: Animal cell cultures have been and are used to generate valuable products based on their own genetic information or due to genes transferred into them using recombinant DNA technology. On the other hand, biotechnology approaches are used either to rapidly multiply animals or desired genotypes or to introduce specefic alterations in their genotypes to achieve certain userful goals. To achieve the latter more efficiently as well as to assist in conventional breeding efforts, the entire genomes of animals are being characterised using biotechnology tools. These activities have been arbitarily grouped under animal biotechnology since they either utilize animal cells to generate products or apply biotechnological tools to enhance the usefulness of animals to human welfare.
Cell and Culture Products. Animal cell cultures are used to produce virus vaccines, as well as a variety of useful biochemical which are mainly high molecular weight proteins like enzymes hormones, cellular biochemical like interferons, and immunobiogical compounds including monoclonal antibodies. Animal cells are also good hosts for the expression of recombinant DNA molecules and a number of commercial products have been are being developed initially, virus vaccines were the dominant commercial products from cell culture but at present monoclonal antibody production is the chief commercial activity. It is expected that recombinant proteins would become the prime product from cell cultures in the near future. Transplantable tissues and organs are another very valuable product from cell cultures. Artificial skins are already in use for graftin in burn and other patiens, and efforts are focussed on developing transplantable cartilage and other tissues. A greater detail of cell culture products is provided under the following heads (1) vaccines (2) interferon (3) monoclonal antibodies (4) hybrid antibodies and (5) recombinant proteins.

Cloning

Cells derived from a single cells through mitosis constitute a clone and process of obtaining clonies is called cloning. (A sexual progeny of single individual make up a clone). In simple terms cloning consists of trypsinisation of a monolayer culture to prepare a cell suspension 3-4 dilution steps to achieve suitable cell density (10-200cells/ml) and seeding in petri dishes or flasks or multiwell dishes the culture vessel are incubated for 1-3 weeks with a medium change after 1 week by this time colonies will develop.
Colonies may be isolated (1) directly from multiwell dishes by tryupsinisation of usually such wells which contained only one cell the start or which have single distinct colony originating from single cell lying away from other nondividing from petriplates is done by using cloning ringes which are placed around the desired colonies after the medium is poured. The cell colony within each porclain teflon well of multiwell plate or in a flask. Alternatively (3) the desired colony may shielded and the remaining colonies are irridiated by a lethal dose (3,000rads) The protected colony is trypsinised and the cells are cloned in the same plate the irridiated cells serving as a feeder layer.
plating efficiency (per cent of cells forming colonies of continuous cell lines is generally 10% or higher, while that of finite cell lines may be quite low say 0.5- 5% or even zero several approaches have been used to enhance the plating efficiency e.g (1) use of a rich medium (2) use of serum espacially foetal calf serum in the medium (3) using conditioned medium (4) use of feeder layer (5) addition of hormones like insulin dexamethasone etc. (6) providing intermediate metabolites like keto acids, nucleosides etc.
A feeder layer is a layer of cells which have been treated to prevent their growth and ultimately cause their death these cells however, provied the necessary metabolites to enahance the plating embryo fibroblast from primary culture and reseeding the cells at density. When the monolayer reaches 50% confluence is covered by the monolayer the cells are either treated with mitomycin overnight or are irridiated with and X-rays. After treatemnet the cells are incubated for 24 hours in fresh medium trypsiised and reseded at and incubated for 24-48 hours. This yields the feeder layer on which cells to be cloned are seeded incubated and desired colonies isolated. Feeder layer may be established using homologous cells but is preferable to use heterologous cells for easy deetection of accidental cross contamination in the isolated clones.
Cloning is used to (1) obtain homogeneous cell lines from heterogeneous cell (2) to isolate biochemical mutants and (3) cell strains with marker chromosomes and (4) to develop hybridoma clones. Cloning is generally applied to continuous cell lines but often their clones become considerable heterogenous by the time they are sufficient multiplied for use. The problem with finite cell lines is that of life span, by the time the clone is suffi

Colony Hybridization

This techniquesis used to identify those bacterial colonies in a plate which contain a specific DNA sequence. These bacterial colonies are obtained from bacterial cells into which this sequence was introduced through genetic engineering. and the given sequence is represented by the probe used in the hybridizaion experiment the procedure for colony hybridization is briefly described below.
(!) The bacterial cells subjected to transformation areplated onto a suitable agar plate this is the master plate.
(2) The colonies of master plate are replica plated onto a nitrocellulose filter membrane placed on agar medium. For replica a block of wood or cork, of suitable diameter for the master plate, is covered with velvet cloth. This block sterlized and the lowered into the master plate till the velvet touches all the colonies the block is withdrawn and gently lowered onto the nitrocellulose filter so that bacterial cells sticking onto the velvet are transferred onto the filter. THe master plate is retained intact for later use. A reference point is marked both on the master plate and the on replica plate to facilitate later comparisons.
(3) After the colonies appear, the filter is removed from the agar plate and treated with alkali to lyse the bacterial cells. This also denature theDNA released fromthese cells.
(4) The filter is treated with proteinase K to digest and remove the proteins the denatured DNA remains bound to the filter.
(5) The filter is now back at tofix the DNA this yields the DNA print of bacterial colonies in the same relative positions as those of the colonies themselves in the master plate.

multiplication expression and integrations of the DNA insert in host genome.

Once the clone containing the desired DNA insert is identified.It is multiplied in E.coli to obtain sufficient number of copies to be used in one or more of the following ways. (1) It canbe used for a structural analysis of the insert e.g DNA sequencing, chromosome walking etc. (2) IT may be introduced into a bacterium like it subtilis for production of the protein encoded by the insert since this host secretes protein into the medium which allows easy purification (3) It can be introduced into a eukaryotic host. e.g yeast, animal cells plants etc. either to investigate the function of the insert or (4) to integrate it into the host genome to achieve one of many diverse objective the aims, techniques and the achievements.

Introduction of the vector into a suitable host

The recombinant vector is constructed in vitro it is then generally introduced into E.coli (1) select the recombinant from the unchanged vector, (2) to obtain many copies of the recombinant vector or the DNA insert, or (3) to express the insert in E.coli itself, purified recombinant vector may subsequently be introduced into another bacterium, e.g Bacillus subtitilis, streptomyces etc. yeast, higher plants or animals. The various approaches for introducing recombinant vectors briefly described while considering the various types of vectors.
Increased competence of E. coli by CaCl2 treatment. E.coli cells are generally poorly accessible to DNA molecules. But treatment with CaCl2 makes them permeable to DNA the process involved is poorly understood growing E.coli cells are isolated and suspended in 50mM cacl2 at a concentration of 10*8 cells the cells may be incubated for 12-24 hours to increase the frequency of transformation. The recombinant vector is then added efficient transformation takes transformed clones. The frequency of transformed cells is per mg of plasmid DNA this is about one transformation per 10,000 plasmid molecules. This frequency can be further improved by using special E.coli strains e.g. SK 1590, SK 1592, etc.
Infection by vectors packaged as virions. Alternatively, those vectors that have the phage cos sequences e.g cosmids, phasmids and vectors, are generally packaged in vitro into specially produced empty phage haeads and complete particles are constituted. These phage particles are used to infect E.coli cells. These vectors can also be used to transform E.coli cells directly as naked DNA, using the CaCl2 technique. Generally infection by phage particles containing DNA insert is far more efficient than direct transformation. For example, the frequency of infection by recombinant phage vectors packaged in phage particles is up to 10*8 plaques of DNA while it is less than 10*3 DNA when the recombinant vector is used for transformation by the CaCl2 technique. The infected transformed bacterial cells are spread on lawn of susceptible cells. Where celar areas or plaques develop in the lawn. Plaques containing the recombinant vector identified and the phage particles collected from such plaques provide the purified vector.

Vectors For Plants

Plants cells do not contain any plasmid. BUt two plasmids called pTi and pRi, and present in the bacteria Agrobacterium tumefaciens and A. rhizogens respectively provide a naturally occuring transformation system. These plasmids transfer a part their DNA called T-DNA into the genomes of most dicot and some monocot plants These plasmids, especially the Ti plasmid, have been used to develop a variety of vectors. In addition, genomes of many plant viruses are being developed in vectors. The purpose of plant vectors is almost always a stable transformation ordinarily in the form of integration in plant genomes. But in the case of virus vectors, the objective is to produce large quantities of the protein encoded by DNA insert.

SV 40 Vectors

It is a spherical virus with a circular double stranded 5243 bp chromosome which encodes 5 proteins viz small T large T bothe early protein VP1 VP2 and VP3 (vp virion protein) has an origin or replication about 80 bp and is completed with hostones toform chromatin large T is essential for viral replication when VP1 VP2 VP3 form the viral capsid. In laboratory, it is multipliedincultured kidney cells of African green monkey, infected cells live after 4 days releasing upto 10 *5 virions Sv40 genome has been used to develop mainly 3 types of vector (1)transducing vectors, (2) plasmid vectors and (3) transforming vectors.
(1) SV 40 transducing Vectors. There vectors produce viral particles infecting monkey cells. They must have these 3 features (1) the sv 40 origin include the surrounding region containing the transcriptional regulatory signals i.e regions at which slicking and polyadenylation (2) size including the of the DNA insert between 3900 bp and 3500 bp for packaging into virons and (3) genes encoding large T, VP1 VP2 and Vp3. This leaves very little rooms for DNA inserts.
(2) Large replacement Vectors. The regions encoding VP1 Vp2 and Vp3 must be replaced in the vectors by DNA insert such a vector is called late region replacement vector. e.g SVGT-5. A vector of this type is used for infection of host cells in conjunction with another virus, called helper virus, which has the VP1 VP2 and VP3 genes intact but has defective large-T gene. In this casem onl those host cells that are infected by both the vectors and the helper virus will lyse and produce virions isnce cells infected by either the vectors the helper alone will not support packaging or replication . This feature is very useful since all the plaques formed or monkey cell monolayers contain the vector.
(3) Early replacement vectors. Alternatively the essential genes missing from the vector may be present within the genome of host cells. FOr example, COs (cv-1, origin of SV 40 CV-1 is a monkey cell line cell line of African green monkey kidney cell cultures contains in its genome the gene for large T of SV40. Therefore, a vector having the origin of replication and genes for VP1 VP2 and Vp3 will replicate and produce virions in COA cell line cells. In such a case, no helper virus is required. Since in such a vector the early genes large T are replaced by the DNA insert, is is called early region replacement vector.
SV 40 plasmid Vectors. These vectors replicate in monkey cells but do not get packaged into virions they contain the origin of replication and the large T encoding gene (large T gene is not necessary for multiplication in COA cells. Obviously, there is not size limit on such vectors, and some of them are E. coli and monkey shuttle vectors e.g pSV2, pSV3 etc. These vector produced high copy number per cell. The shuttle vectors are used to propagate the recombinant vector in E.coli which are then introduced into monkey cells to study the expression of DNA inserts.

Non-replicating vectors

These vectors do not replicate. They only serve as vehicles of transfecting DNAs which may become intetrated into the host DNA such vectors are therefore also called passive transfecting vectors. These vectors are generally shuttle vectors. THey are first cloned in E. coli to isolate recombinant vectors and then used to transfect various mammalian cells since they need not be restricted to monkey cells in view of their lack of replication. The Sv40 segments used tin these vectors are generally the transcription regulatory sequences and the polyadenylation sites. Permanent transformants are selected on the basis of selectable markers present in the vector.
The selectable marker need not be convalently linked to the DNA insert or the transfecting DNA. Even when two seperate DNA fragments containing seperate each are mixed and used for for transfection more than 50% of the permanently transfected cells contain both the genes usually integrated side by side the two DNA fragments tend to become joined after entering the animal cells which is the reason for their co-transduction i.e integration of the two genes together in the genome.

Bovine Papillomavirus (BPV) vectors

Pappilomavirus belong to papova virus class and causes wanrts. They have ciruclar 8 kb genome organised in nucleosomes. Th ebovine papillomavirus replicate as a stable plasmid in rodent and many bovine cells and the cells are killed. The viral genome transforms cells which behave like tumor cells and from piled up colonies of cells instead of the typical monolayer. THe transformed state is due to the genes present in the transforming region (about 5500 bp of the virus genome. The virus genome is generally used to produce shuttle vectors by using the transforming region of viral genome.
Eukaryotic DNA segments are first cloned in E. coli to select recombinant vectors. Then the E. coli plasmid say pBR 322 is deleted from the vectors, and the linear recombinant vector is introduced into animal cells the vector becomes circular and replicates as plasmid. The E.coli neo gene may be included within the vector this allows easy selection of transfected cells by culturing them on medium containing the aminoglycoside G-418.

Retrovirus Vectors

REctroviruses have single stranded RNA genomes which are transcribed by reverse transcriptase in a DNA double strand copy inside the host cells. THe DNA copy integrates into the host genome to become ap provirus which causes permanent transfection of the cells. The provirus genome is transcribed and expressed virions are formed and extruded into the medium. Retroviral vectors haveb the folowing three features. (1) the vector has viral sequences for replication, gene expression and packaging sequences (2) DNA inserts may either replace or be locaed in the nonessential coding region of the viral genome. (3) The vector and the recombinant vectors are packaged into virions and used as transducing phages (4) The viral proteins are usually provided by a helper virus or a provirus. (5) DNA copies of the retrovirus genome are used as vectors. generally as shuttle vectors.
A typical vector has the following (1) pBR 322 ori and a selectable marker (2) retroviral 5'LTR and 3" LTR (3) R,U5, U3, p and pu encoding sequences (involved in reverse transcription ) (4) sequences necessary for packaging into virions (5) sequences needed for splicing to produce functional mRNA for envelope protein synthesis, and (6) atleast one unique restriction site for insertion of DNA fragment without interrupting any of the essential sequences.
THe recombinant shuttle vector is constructed and first cloned in E.coli it is then isolated and tintroduced into animal cells where the entire vector, expect the E. coli plasmid dequence, is transcribed. This transcript contain RNA copy of the DNA insert, and is packaged into virions and is infective on animal cells. Since the vector does not encode the viral capsid proteins, it has to be supplied by a helper virus or provirus. If the provirus or helper virus lacks the sequence, it genome is not packed into virions. As a result, all the irions recovered from the medium contain the recombinant vectors. The recombinant vector so recovered can be used in further experiment. The recombinant vector can integrate as provirus into the host genome the transfected cells can be easily selected either as piled up colonies of cells if the vector retains an oncogene or due to the expression of an E. coli gene e.g gene neo conferring resistance to the drug G-418.

Yeast Vectors

Yeast, Saaccharomyces cerevisiae, is an eukaryotic with 34 chromosomes. it reproduces sexyally as well as asexually be budding. In suspension cultures it grows as single cells with cell doubling time of 1.5 to 2.5 hrs but on agar plates cells produce colonies. Its haploid DNA content is only times that E. coli and its genetics hasbeen extensively studied. Yeast viruses are not known only a single yeast plasmid has been discovered which has been used to contrast some useful vectors. The tough polysaccharide wall of yeast is an effective barrier to DNA molecules. Therefore, yeast cell wall is enzymatically digested to produced spheroplasts which can take up DNA following treatment with cacl2 walls regenerate the specific media.

Shuttle Vector

These vectors have been designed to replicate in cells of two different species therefore they contain two origins of replication, one specific for each host species as well as those genes necessary for their replication and not provided by the host cells. Thesevectors are created by recombinant techniques. Some of them can be grown in two different prokaryotic species, while others can propagate in prokaryote species, usually E. coli and a eukaryotic one e.g yeast, plants, animals Since they vectors can be grown in one host and then moved into another without any extra manipulation they are called shuttle vectors.
A shuttle vector designed to replicate in E.coli and streptomyces has been constructed as follows (1) the modules for DNA replication in streptomyces and mtehylenomycin a resistance are derived from a streptomyces plasmid, and (2) the replication module or maintenance in E. coli and a gene for antibiotic resistance are taken from anE.coli plasmid. This shuttle vector allows the initial cloning of streptomyces DNA inserts in E.coli and their subsequent functional test in streptomyces shuttle vectors have been designed to specifically satisfy this need i.e the initial cloning of DNA inserts in E.coli and subsequent functional test in the species to which the DNA inserts belong. Most of the eukaryotic vectors are in fact shuttle vectors.

Phasmid Vectors

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from
prokaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.

Cosmid Vectors

Cosmid are essentially plasmids that contain 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 terminaase so that under appropriate condition they are packaged in vitro into empty phage particles cosmid has (1) replication origin (2) unique restriction sites and (3) selecetable markers from the plasmid therefore selection stratgy for obtaining the recombinant vectors 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 precusors a for packaging in particles. The procedure selects for long DNA insert since for packaging the distance between two cos sites must between 38 and 50 kb. Cosmid can accomodate upto 45 bp 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 particles which infect host cells, which many fold more efficient than plasmid transformation (3) Selection for recombinant vector 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.

Selection of Recombinant Vector

It may be pointed out that when an experiment is performed to insert a DNA fragment into a
vector, two types of vectors molecules are obtained (1) many vector molecules will contain the DNA insert but, (2) many others well contain only the vector sequences. This mixture of vector molecules is used for transformation of host cell. (1) Some host cells will receive the recombinant vector, (2) some others will contain the normal unaltered vector, while (3) the majority of them will contain no vector, i.e will not be transformed. In a cloning experiment it is critical to effectively select for the low frequency of cells transformed by the recombinant vector form among the cells containing the un altered vector and the non transformed cells.
Selection of host cells transformed by the recombinant vector is easily achieved by placing two selectable markers, e.g. antibiotics resistance gens, such as a ampicillin resistance and tetracycline
resistance in the vector. The DNA insert is integrated within one of the two selectable rmarkers. If the DNA insert is integrated with in the ampicillin resistance gene, the cells containing recombinant vector will be resistant to tetracycline but sensitive to amipicillin. In contrast, non transformed cells will be sensitive to both the antibiotics, while that containing the unaltered vector will is resistant to both. Therefore, following transformation with the above recombinant vectors cells are plated on a tetracycline supplemented medium this eliminates the nontransformed cells. The remaining colonies are not replica plated on ampicillin vector, and are isolated from the master plate. Further, transformed cells tend to lost the recombinant vector, since cells lacking such vectors divide much faster. The use of a vector having two selectable markers allow the maintenance of cells containing the recombinant vector on antibiotic medium which eliminates the vector free cells produced during culture.

PCS101

This earliest plasmid vector contains the replication module for replication in E.coli gene for resistance tetracycline and single recognition sites for restriction. Insertion of DNA insert into the ECoR1 site leaves the gene in act and functional as a result E. cells transformed by psc101 become the DNA insert or a recombinant one on the other hand insertion of DNA fragment into the Hind111 Bam H1 or Sal sites disrupts the gene and makes it nonfunctional. Therefore, cells transformed by such a recombinant are sensitive to tetracycline and hence can be easily distinguished from those containing the non recombinant plasmid which are resistant to the antibiotics.
But non transformed cells too are tetracycline sensitive hence they can not be separated from those having the recombinant.
Clearly pSC101 does not permit a direct selection of cells containing the recombinant vector. In addition, it contains unnecessary DNA, and has stringent regulation of replication. Subsequently, ser=veral novel plasmid vector were designed to over come these deficiencies.

PUC 7

It is a derivatie of PBR322 and is much smaller it has all the essential parts of Pbr 322 (1) ampicillin resistance gene and (2) Col El origin. The second scorable marker is due to E. coli gene lacZa encoding the or fragment.of B-galactosidase the enzyme that hydrolyses galactose. The E.coli strains e.g. JM103, JM 109, used as hosts for the pUC series vectors have laacZa deleted from their lac operan. When pUC enters such an E. coli cell, the host genome and the plasmid encode for different parts of the B-galactosidase enzyme which interact with each other to produce the active enzyme enabling these cells to hydrolyse galactose B hydrolase also hydrolyses X-gal (5-Bromo-4-chloro-3-indolyl-B-D galactoside) to yield a blue dye. Therefore appropriate lacZ E. coli cells transformed by the pUC sectors behave as lacZ+ and produce blue coloured colonies on a X-gel containing medium. A polylinker sequenced by itself does not intercfere with lac Za expression, but when a DNA insert is placed within lac Za expression is prevented.
The unique restriction sites used for integration of DNA inserts into pUC vectors interrput the lac Za fragment so that appropriate E. coli cells possessiong recombinant pUC vectors are B-galactosidase deficient and as a result produce while colonies on X-gel medium. Therefore, appropriate E. coli cells transformed with pUC vectors are first grown on ampicillin containing medium to eliminate medium. the while colonies are selected as they contain the recombinanat vector blue colonies will contain the unaltered vector The other vectors in pUC series are pUC8 pUC 9 pUC 12 pUC 13 etc.

Biotechnology in industry

As technology advances, the many roles biotech plays in our lives increases. Biotechnological advances can be found in nearly all sectors of industry today. There are, of course, the obvious medical, pharmaceutical and food industries. Biotechnology is being used to determine cause and effect of various diseases and are used in the production of drugs. The production of foods is enhanced by biotechnological advances that improve crop yields, introduce in-situ insect resistance and provide new ways of food preservation. Technological advances also include biodegradable packaging and built-in bio-indicators of food contamination. In the environmental sector, biotech has played a role in remediation of contaminated land, water and air, pest control, treatment of industrial effluents and emissions, and acid mine drainage.
Biotechnology is technology based on biology, especially when used in agriculture, food science, and medicine. The UN Convention on Biological Diversity has come up with one of many definitions of biotechnology: "Biotechnology means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."

Traditional pharmaceutical drugs are small chemicals molecules that treat the symptoms of a disease or illness - one molecule directed at a single target. Biopharmaceuticals are large biological molecules known as proteins and these target the underlying mechanisms and pathways of a malady; it is a relatively young industry. They can deal with targets in humans that are not accessible with traditional medicines. A patient typically is dosed with a small molecule via a tablet while a large molecule is typically injected. Small molecules are manufactured by chemistry but large molecules are created by living cells: for example, - bacteria cells, yeast cell,animal cells.

E. coli vectors

Bacteria are the bests of choice for DNA cloning among them, E.coli occupies a prominent posion since cloning and isolation DNA inserts for structural analysis is the easiest in this best. Therefore, the initial cloning experiments are generally carried out in E.coli. The E.coli strain k 12 is the most commonly used. It has several substains, e.g. c600 HB101 each of which has some specific features important in cloning. For example, the substain RRi hasm in addition to certain other features, the mutation hsdr which inactivates the restriction enzyme endogous to E coli k12, this minimises the degradation of recombinant DNA introduced into it.
Properties of good host: A good host should have the following features, (1) is easy to transform (2) supports the replication of recombinant DNA, (3) is free from elements that interfere with replication of recombinant DNA,(4) lacks active restriction enzymes, e.g. E. coli k12 substrain HB 101, (5) does not have methylases since these enzymes would to useful restriction enzymes, and (6) is deficient in normal recombination function so that the DNA insert is not altered by recombination events.
E. coli supports several types of vectors, some natural some constructed, which can be grouped as follows: (1) plasmids, (2) bacteriophages (3) cosmids, (4) plasmids, and (5) shuttle vectors
Plasmids.
A plasmid is a DNA molecule, other than the bacterial chromosome, that is capable of independent replication and transmission. Plasmid are circular and may exits either independent of or may become intergrated into the bacterial chromosome gernally they are not essential for the host cell except under spefcific environments. There are severaltyupes of bacterial plasmids, but the three widely studied types are F plasmids (2) R plasmids and (3) Col plasmids the protein that kill sensitive E. coli cells, they also carry genes that provide immunity to the particular through conjugation, and as a result spread rapidly among the bacterial cells of a population e.g F plasmids, many R plasmids and some Col plasmids, or nonconjugative ( do not mediate DNA transfer through conjugation) e.g many plasmids and most Col plasmids.
stringent and relaxed replication. Each plasmid is maintained in the bacterial cell at a characteristic copy number mainly due to its replication control system. In this respect the plasmids are of two types (1) single copy and (2) multiple copy plasmids. The replication control of single copy plasmids is the same as that of their bacterial host cells so that they replicate and segregate with the bacterial chromosome this is called stringent replication. In contrast, the replication control of multi copy plasmids is different from that of their bacterial host genome so that they undergo more than one replication for each replication of their host genome this is referred to as relaxed replication.
Modular organization. Plasmids may visualized as constructed from modular DNA segments. A module may be regarded as a DNA segment or sequence performing a specific function each module may contain one or more genes.

Cloning and Expression Vectors

All Vectors used for propagation of DNA insers in a suitable host are called cloning vectors. But when a vector is designed for the expression of production of the protein sepcified by the DNA insert, itis termed as expression vector. As a rule such vectors contian atleast the regulatory sequences, promoters, operators ribosomal binding sites ets, having optimum function in the chosen host. It is desirable that all cloning vectors have relaed replication control so that they can produce multiple copies per host cell.
When an eukaryotic gene to be expressed i a prokaryote, the eukaryotic coding sequence has to be placed after prokaryotic promoter and ribosome buildin site since the regulatory sequences of eukaryotic are not recognised in prokaryotes in addition, eukaryotic genes as a rule, contain introns presend within their coding regions. These introns must be removed to enable the proper expression of eukaryotic genes since prokaryotes lack the machinery needed for their removal from the RNA transcripts. When eukaryotic genes are issolated as Cdna they are intron free and hence, suitable for expression in prokaryotes.
Several strategies have been attempted for the construction of expression vectors using regulatory sequences of the appropriate hosts. These approaches may be grouped into the following two broad categories.
1. Construction of vectors allowing the synthesis of fusion proteins comprising amino acids coded by a sequence in the vector and those encoded by the DNA (translational fusion).
2. Development of vectors permitting the synthesis of pure proteins encoded exclusively by the DNA inserts (transcriptional fusion)
Examples of the first strategy producing fusion proteins are the expressions insulin fat growth hormone, structural protein VPI of foot and mouth disease virus, human growth hormones etc. Some examples of the second approach producing unique proteins are rabbit B globin, small antigen of SV 40, human fibroblast interferon, human human IGF-1 protein. It may be pointed out that the undesired amino acids encoded by the vector sequence in case of translational fusion must be removed from the fusion proteins by a suitable chemical cleavage.
Several other problems are faced when eukaryotic genes are expressed in a prokaryotic system, e.g removal of signal sequences from precusor proteins to obtain active mature protein molecules. Various strategies are being rapidly devised to effectively overcome these problems.

Vectors

A Vector is a DNA molecule that has the ability to replicate in an appropriate hostcellm and into which the DNA fragment to be cloned called DNA insert is intergrated fro cloning. Therefore, vector must have an origin of DNA replication that functions in the host cell. ANy extra chromosomal small genome e.g plasmid phage and virus, may be used as a vector.
Properties of A good vector
A good vector must have the following properties.
1. It should be able to replicate autonomously, when the objective of cloning it to obtain a large number of copies of the DNA insert, the vector replicon must be under relaaxed control so that if can generate multiple copies of itself an a single host cell.
2. IT should be easy to isolate and purify.
3. it should be easily introduced into the host cells, transformation of the host with the vector should be easy.
4. The vector should have suitable marker genes that aloow easy detection or selection of the transformed host cell.
5. When the objective is gene transfer, it should have the ability to intergrate either itself or the DNA insert it carries into the genome of the host cell.
6. The cells transformed with the vector molecules containing the DNA insert recombinant or chimaeric vector should be identifiable or selectable from those transformed by the vector molecules only.
7. A vector should contain unique target sites for as many restriction enzymes as possible into which the DNA insert can be integrated without disruptin an essential function.
8. When expression of the DNA insert is desiredm the vector should contain atleast suitable control elements, e.g promoter operator and ribosome binding sites several other features may also be important.
It should be kept in mind that (1) the DNA molecules used as vectors have coevolved with their specific natural host species, and hence are adapted to function well in them and in their closely related species. Therefore, the choice of vector depends largely on the host species into which the DNA insert or gene is to be cloned. In addition, (2) most naturally occuring vectors do not have all the required functions therefore, useful vectors have been created by joining together segments performing specific functions from two or more natural entities.

Isolation of the desired gene

Comparison of gene-expression patterns between cells and/or tissues facilitates the identification of molecules activated by a particular physiological or pharmacological treatment. The use of gene-expression profiling is particularly important in neuroscience, clinical science, stem cell biology, and metagenomic analysis. In many cases, however, the amount of specimen tissue available is limited, allowing only small amounts of mRNA to be obtained. As such, amplification of the isolated RNA is obligatory to obtain the microgram amounts of RNA required for microarray analysis or cDNA library preparation. Without amplification, such amounts of RNA would be obtainable only from millions of cells. Although the polymerase chain reaction (PCR) is a powerful method for amplifying a single target DNA, the exponential amplification that can be achieved using multiple targets (from mixtures of DNA fragments or mRNA molecules) often produces a biased sample, since cDNAs of differing lengths and composition are amplified with differing efficiencies.

Construction of Genomic library.

For preparation of genomic library, the total genomic DNA of an organism is extracted. The DNA is broken into fragments of appropriate size either by mechanical shearing this generates blunt ended fragments or sonication, or by using a suitable restriction endonuclease for partial digestion of the DNA complete digestion is avoided since it generates fragments that are too heterogenous in size For partial digestion, restriction enzymes having 4-base (thxameric) target sites, This is because a given 4-base recognition site is expected to occur every (= 4096) base pairs it is assumed here that the arrangement of the 4 bases in DNA molecules is random. Therefore, the fragments produced in partial digests with enzymes havaing 4 base recognition sites are molre likely to be of appropriate size for cloning than those generated by enzymes having 6 base recognition sites. Single or mixed digestion with the enzyme genomic libraries. The use of restrictin enzymes has the advantage that the same set of fragments are obtained from a DNA each time a specific enzyme is used, and many of the enzymes produce cohesive ends.
The partial digests of genomic DNA are subjected to agarose gel electrophoresis or sucrose gradient centrifugation for separation from the mixture of fragments of appropriate size.

Genomic Library

A genomic Library is collection of plasmid clones or phage lyates conianing recombinant DNA molecules so that the sum total of DNA inserts in this collection, ideallym repsresents the entire genome of the concerned organism. However, inspite of all the cre taken in the production of genomic libraries, certian DNA gragments should be expected to be under or over represented or even missing. The possible reasons for this may be that certain fragments code for a toxic product, or might replicate slowly or might have been altered by recombinational events during clonign in addition endonuclease cleavage sites are often not recongnised equally well. Certain DNA fragments may therefore never appear in partial digests by a restriction endonuclease used for the constriuction of genomic libraries.

Steps in gene cloning

The entire procedure of cloning or recombinant DNA technology may be classified must the following five steps for the convenience in description and on the basis of the chief activity preformed.
1. Identification and isolation of the desired gene or DNA fragment to be cloned.
2. Insertion of the isolated gen in a suitable vector.
3. Introduction of this vector into a suitable organism or cell called host (transformation).
4. Selection of the transformed host cell, and
5. Multiplication /expression/ integration followed by expression of the introduced gene in the host.
A brief description of these steps is given in the following sections.

Types of cloning

When the media report on cloning in the news, they are usually talking about only one type called reproductive cloning. There are different types of cloning however, and cloning technologies can be used for other purposes besides producing the genetic twin of another organism. A basic understanding of the different types of cloning is key to taking an informed stance on current public policy issues and making the best possible personal decisions. The following three types of cloning technologies will be discussed: (1) recombinant DNA technology or DNA cloning, (2) reproductive cloning, and (3) therapeutic cloning.


Recombinant DNA Technology or DNA Cloning

The terms "recombinant DNA technology," "DNA cloning," "molecular cloning," and "gene cloning" all refer to the same process: the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.


Scientists studying a particular gene often use bacterial plasmids to generate multiple copies of the same gene. Plasmids are self-replicating extra-chromosomal circular DNA molecules, distinct from the normal bacterial genome (see image to the right). Plasmids and other types of cloning vectors were used by Human Genome Project researchers to copy genes and other pieces of chromosomes to generate enough identical material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA. See a diagram depicting this process.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial plasmids, some other cloning vectors include viruses, bacteria artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Cosmids are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA and can be packaged in lambda phage particles for infection into E. coli cells. BACs utilize the naturally occurring F-factor plasmid found in E. coli to carry 100- to 300-kb DNA inserts. A YAC is a functional chromosome derived from yeast that can carry up to 1 MB of foreign DNA. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

Reproductive Cloning

Celebrity Sheep Died at Age 6

Dolly, the first mammal to be cloned from adult DNA, was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. The unnamed sheep from which Dolly was cloned had died several years prior to her creation. Dolly was a mother to six lambs, bred the old-fashioned way.

Image credit: Roslin Institute Image Library

Reproductive cloning is a technology used to generate an animal that has the same nuclear DNA as another currently or previously existing animal. Dolly was created by reproductive cloning technology. In a process called "somatic cell nuclear transfer" (SCNT), scientists transfer genetic material from the nucleus of a donor adult cell to an egg whose nucleus, and thus its genetic material, has been removed. The reconstructed egg containing the DNA from a donor cell must be treated with chemicals or electric current in order to stimulate cell division. Once the cloned embryo reaches a suitable stage, it is transferred to the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is not truly an identical clone of the donor animal. Only the clone's chromosomal or nuclear DNA is the same as the donor. Some of the clone's genetic materials come from the mitochondria in the cytoplasm of the enucleated egg. Mitochondria, which are organelles that serve as power sources to the cell, contain their own short segments of DNA. Acquired mutations in mitochondrial DNA are believed to play an important role in the aging process.

Dolly's success is truly remarkable because it proved that the genetic material from a specialized adult cell, such as an udder cell programmed to express only those genes needed by udder cells, could be reprogrammed to generate an entire new organism. Before this demonstration, scientists believed that once a cell became specialized as a liver, heart, udder, bone, or any other type of cell, the change was permanent and other unneeded genes in the cell would become inactive. Some scientists believe that errors or incompleteness in the reprogramming process cause the high rates of death, deformity, and disability observed among animal clones.


Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of human embryos for use in research. The goal of this process is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Stem cells are important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Stem cells are extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryo, which raises a variety of ethical concerns. Many researchers hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases. See more on the potential use of cloning in organ transplants.

In November 2001, scientists from Advanced Cell Technologies (ACT), a biotechnology company in Massachusetts, announced that they had cloned the first human embryos for the purpose of advancing therapeutic research. To do this, they collected eggs from women's ovaries and then removed the genetic material from these eggs with a needle less than 2/10,000th of an inch wide. A skin cell was inserted inside the enucleated egg to serve as a new nucleus. The egg began to divide after it was stimulated with a chemical called ionomycin. The results were limited in success. Although this process was carried out with eight eggs, only three began dividing, and only one was able to divide into six cells before stopping.

Cloning

Cells derived from a single cell through tmitosis constitute a clone and the process of obtaining clones is called cloning. In simple term cloning consists of trypsinisation of a monl=olayer culture to prepare a cell suspension, 3-4 dilution steps to achieve a suitable cell density (10- 200 cell/ml) and seeding in petri dishes or flasks or multi well dishes The culture vessel are incubated for 1-3 weeks with a medium change after 1 week by this time colonies will develop.
Colonies a may be isolated (1) Directly from multiwell dishes by trypsinisation of usually such wells which contained only one cell at the start, or which have single distinct colony origination from a single cell lying away from other nondividing cell or cells. This is confirmed by microscopic observation (2) Isolation of clones from petriplates is done by using cloning rings which ar eplaced around the desired colonies after the medium is poured. The cell colony within each porcelain teflon or stainless steel ring is trypsinized, cell are suspended in medium and seeded in a shielded and the remaining colonies are irridiated by a lethal dose The protected colony is trypsinised and the cells are cloned in the same plate, the irridiated cells serving as a feeder layer.

Plating efficiency lines is generally 10% or higher, wehile that of finiite cell lines may be quite low say 0.5-5% or even zero. Several approaches have been used to enhance the planting efficiency e.g (1) use of rich medium, (2) use of serum, especially foetal cell serum, in the medium, (3) using conditioned medium grwon for a period of time (4) use of feeder layer, (5) addition of hormones like insulin, dexamethason etc, (6) providing intermediate metabolited like keto acids nucleosides etc.

GENE THERAPY

Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.

The history of human gene therapy is, however, not a particularly happy one. The effect of introducing a gene into cells rarely promotes more than small transient relief from the symptoms of the disease being treated. Worse still, there have been highly publicized cases where gene therapy trial patients have suffered as a consequence of the treatment itself. For example, in 1999 an 18-year-old gene therapy trial volunteer from Philadelphia died following a gene therapy trial. In addition, one of the few success stories of human gene therapy—the treatment of severe combined immune deficiency, X-SCID—has turned out to have unforeseen consequences. Bone marrow cells were taken from patients suffering from this disease and treated with a virus to introduce a functional copy of the defective gene. When the modified bone marrow cells were returned to patients, their immune systems were functional once more. However, some patients treated this way subsequently developed leukaemia, which most likely arises as a result of random insertion of a section of DNA into the human genome with the consequent disruption of nearby gene function.

RESTRICTIN ENDONUCLEASE

Endonuclease are enzymes that produce internal cuts, called cleavage, in DNA molecules. Many endonucleases cleave DNA molecules at random sites. But a class of endonucleases cleaves DNA only within or near those sites which have specific base sequences, such endonucleases are known a restrictin endonucleases, and the sites recognised by them are called recognition sequences and the recognition sequences are different and specific for the different restriction endonucleases of restriction enzymes. Restiction enzymes were discovered due to and named after the phenomenon of host restriction of bacterial phages. When a phase DNA released from one bacterial stain (Say E. coli strain such a phage is designated is used to infect another strain (say E. coli strain K ) the effieicney of phage growth on the latter (strain K) is only a very small fraction of the efficiency on the former strain C some phages that survive grow normally on the second strain, and are now referred to as K these phage particles infect strain K with the same efficiency restrict the growth of the phage to teh strain concerned. This restriction on phage host range is due to the presence of restrtiction enzyme.

Recombinant DNA technology

The recombinant DNA molecule is produced by joinign together two or more DNA sesgments usually originating from different organisms. More specifically a rcombinant DNA molecule is a vector into which the desired DNA fragment has been inserted to enable its cloning in appropriate host. Thisis achieved by using specific enzymes for cutting the DNA (restriction enzyme) into suitable fragments and then for jointing together the appropriate fragments (ligation) In this manner, a recombinant DNA molecule may be produced which contains a gene from one organism joined to regulatory sequences from another organism, such a gene is called chimaeric gene. CLearly, the capability to produced novel gene combination to suit specific needs. Recombinant DNA molecule produced one of three ways (1) to obtain a large number of copies of specific DNA fragments (2) to recover large quantities of the protein produced by the concerned gene (3)

to integrate the gene in question into the chromosome of a target organism where it express itself.

Gene transmission

The high fidelity semiconservative replication of DNA ensures transmission of genes from parents to progeny without change this is the reason for stability of genetically controlled phenotypes over generations. However, a low frequency per gene per generation of changes occur in genes naturally these mutations are the ultimate source of all the heritable variation observed inm living forms. Another source of genetic variation si recombination between genes. In eukaryotes recombination occurs regularly during the meitotic for the formation of gametes involved in sexual reproductin the has been highly successfully exploited by plant and animal breeding programmes. In prokaryotes, on the other hand, recombination occurs when foreign DNA is brought into a cell during any of the folowing three events transformative transduction and conjugation in gransformation DNA is directly takes a by the cell and portion of ti becomes integrated in the cells chromosome a process of recombination. But in case of transduction, the DNA is transferred ffrom one cell into another by a virus it may either be generalized or specialized conjugation on chromosome may be transferred into the reciepent cell often even large plasmids are exchanged. SEgments of the transferred chromosome become into the chormosome of the recipient cell by a process of recombination.

Gene transmission

gene and its function

A gene is the basic structural and functional unit of life. A gene is the part of chromosome and responsible for some character or trait fo an organisms. Gene is made of DNA it is double stranded there are two stranded of DNA which is complementary (A one strand faces T is the other and G in one faces C in the other) and run anti-parallel to each other (the 3' OH of ribose present in one strand and 5' p residue of the other strand are located at the same and of a double helix) DNA replication is highly faithful, is semi conservative and is catalyzed by DNA polymerase.
Genes produce their phenotypic effects by specifying the aminoacid sequences of specific proteins. The nucleotide sequence of one stand of a DNA double helix is used by RNA polymerase as template to generate its complementary copy of RNA this RNA ultimately functions as messenger RNA and the process of its production is called transcription.