Biotechnology PDF

BIOTECHNOLOGY  Biotechnology- Introduction Fusion of biology and technology Term coined by Karl Ereky (1917) Branch of science which deals with the exploitation of biological agents or their components for generating useful products or services It was began with the dis...

BIOTECHNOLOGY  Biotechnology- Introduction Fusion of biology and technology Term coined by Karl Ereky (1917) Branch of science which deals with the exploitation of biological agents or their components for generating useful products or services It was began with the discovery of fermentation and the production of alcoholic beverages Old study includes fermentation, antibiotic production baking, brewing etc. and new studies includes cell culture, cell fusion, genetic engineering etc.  Some defenitions…. It is the application of biological organism, systems or processes It is the multi branch techniques which include the integrated use of biochemistry, microbiology, engineering sciences for achieving industrial application of capabilities of microorganisms, cultured tissue cells and their parts It is the use of biological agents like microorganisms or cellular components It is the technology used for manufacturing the various kinds of useful substances  Branches of biotechnology Medical/red biotechnology -protection of human health by producing recombinant vaccines, monoclonal Ab, drugs, gene therapy, DNA finger printing etc. Industrial/white biotechnology - Production of biochemicals (alcohol to antibiotics), production of enzymes (protease, lipase, amylase). It also includes the remodeling of proteins or enzymes to enhance their efficiency and also for mineral extraction. Environmental biotechnology -for detoxification of industrial effluents, in combating oil spills, for treatment of sewage and for production of biogas  Branches of biotechnology Animal biotechnology - For production of transgenic animals, some proteins and for in vitro fertilization and embryo transfer in human Plant/green biotechnology -production of transgenic plants, producing resistant plants, for germplasm conservation etc. Blue biotechnology - For marine and aquatic application  Scope and importance Its application is mainly in molecular biology and microbiology area It plays an important role in employment, production and productivity, trade, economics and economy, human health and quality of human life In human health protection it means for 1. the production of monoclonal antibodies (used to diagnose many diseases like hepatitis B, cancer etc.) 2. DNA and RNA probes (for diagnosing kala azar, sleeping sickness, malaria etc.) 3. Artificial vaccines 4. Gene therapy-for treating genetic disease 5. DNA finger printing  Scope and importance… In microbiology it means For the production of biochemicals ranging from alcohol to antibiotics For remodeling of protein or enzymes to enhance efficiency For controlling plant diseases and insect pest (virus, fungi and bacteria) For the detoxification of industrial effluents, treatment of sewage and gas production etc. In the sterility persons.. Invitro fertilization and embryo transefer In genetic engineering Production of transegenic animals In agriculture For the rapid and economic clonal multiplication of fruits and forest trees, production of virus free stocks of clonal crops, production of desease and insect resistant verieties.  Fundamentals of Animal cell culture and Hybridoma Technology Cell culture- process of cell growth under controlled condition within a suitable nutrient medium (by Jolly, 1903; Harrison 1907; and Alexis Carrel 1912). It is through enzymatic or mechanical dispersion of tissues or by spontaneous migration of cells from explant Primary cell culture-freshly isolated, here culture is derived directly from tissue through by allowing cell migration from tissue and then these cells were adhere to substrate or by disaggregating the tissue mechanically or enzymatically and produce cell suspension Disaggregation involves- enzymatic digestion by using trypsin, Physical disruption and treatment with chelating agents like EDTA Secondary culture-subculturing from primary culture  Cell lines and types of cell lines Cell lines- permanently formed cell culture which divide indefinitely in a given medium and space Finite cell lines-which may die after several cell culture, ie. they grow through a limited number of cell generations and have a limited life Finite cell line have the following features like- anchorage dependent, contact inhibition and density limitation Continuous cell lines- they are derived from surviving primary cell culture and they may continue to grow indefinitely. Continuous cell lines are obtained either from transformed cell lines or cancerous cells. They divide rapidly and have no contact inhibition and no anchorage dependence and no or reduced density limitation. They have rapid growth and proliferation and shows the altered ploidy due to altered chromosome number Valuable products from cell culture Cell cultures-used for obtaining various useful products biochemical (interferon, interleukins, hormones, enzymes, antibodies etc.) and virus vaccines (polio, mumps, measles, rabies etc.) Large scale cell culture They are the source of valuable products Animal cell products includes high molecular weight proteins like enzymes (asperaginase, collaginase, hyaluronidase, pepsin, rennin, trypsin, tyrosine hydroxylase and urokinase), hormones (LH, FSH, CH and erythropoeitin), animal vaccines, monoclonal Ab, interferon etc. Vaccines FMD vaccine (foot and mouth disease) Others are poliovaccines, bovineleukaemia virus (BLV), mumps, rubella, influenza, measles and rabies Interferons Produced by virus infected cell and give protection to other healthy cell They are a family of proteins (glycoproteins-cytokinins) that are made and secreted by cells of immune system Are antivial agents and can fight against tumours and modulate the immune system to vius, bacteria, cancer and other foreign substances and kill these organism by boosting the immune system response and reducing the growth of cancer cells Interfrons are 3 types- IFNα produced by WBC; IFNβ Produced by fibroblasts and IFNγ produced by stimulated T-cells,so named as immune IF Monoclonal antibodies By in vitro and in vivo mechanisms In vitro is economically viable because for the production of 1 Kg ab, nearly 20,000 mice is required in in vivo Tissue plasminogen activator (t-PA) These are the substance used for dissolving blood clots in patients with heart diseases, thrombic disorders and in acute myocardial infarction Which converts the inactive blood plasminogen to active plasmin and this inturn cleave the insoluble fibrin clot to soluble fibrin fragment which helps for the opening of blood vessels Another products are Factor VIII  Monoclonal antibodies and Hybridoma technology Hybridoma- is a hybrid cell which is obtained by the fusion of B-lymphocytes with tumour cell It has the ability to produce antibodies and the capacity for indefinite growth Hybridoma technology-for obtaining monoclonal antibodies, hybridoma cultured as in vitro or passed through peritoneal cavity of mouse. Monoclonal antibodies-large quantities of plasma cell antibodies produced through HT (Kohler, Milstein and Neils Jerne-1984) Monoclonal antibodies (mAb or moAb) Are homogenous Ab produced by single clone of genetically identical B-cell It is specific to a single antigenic determinants of a single antigen Formed by the fusion of single myeloma cell with a single Ab producing lymphocyte Uses To detect the presence and quantity of Ab specific substance To detect antigen in fixed tissue sections through immunohistochemistry To purify the substance through immunoprecipitation and affinity chromatography Used in serological identification tests To prevent tissue rejection and to make immunotoxins Used to diagnose disease and for blood group classification Used to detect pathogens and the cancer Used to detect diseases through ELISA, agglutination, precipitation etc.  Steps of HT Immunize a rabbit through injection of antigen for the production of specific Ab, due to the proliferation of B-cell Produce tumours in rabbit/mouse Separates the spleen cells and myeloma cells (no Ab synthesis and HGPRT negative-hypoxanthine guanine phosphoribosyl transferase, an enzyme which recycles the building blocks of RNA and DNA) from the culture Fusion of B-lymphocytes with myeloma cells using PEG (poly ethylene glycol) The hybrid cells are grown in HAT (hypoxanthine aminopterine thymidine) medium. They were proliferate in HAT medium since B-lymphocytes makes them HGPRT+ and they have indefinite growth Select desired hybridoma for cloning and Ab production Culture selected hybridoma cells for the production of mAb in large quantities Gene Cloning and DNA Sequencing Genetic Engineering It is the manipulation of genetic material It involve the isolation and combining of DNA fragments. Two molecules of DNA were isolated and cut into small fragments by using enzymes and joined with desired fragment and restored to a cell for its replication and reproduction It requires cloning vectors like plasmid, phage etc. into which desired DNA were inserted and then transfer into host. This process of making of recombinant DNA is known as molecular cloning. A clone is an asexual progeny of a single individual or cell, hence all have the same genotype with that of individual from which they derived Enzymes r DNA Technology Exonuclease Nuclease- degrade the phosphodister bond of nucleic acids Exonuclease degrade at terminal end (5’ or 3’ end). Eg:- Bal 31, exonuclease III Endonuclease Cleave at any point not at end. Eg:- deoxyribonuclease 1 (Dnase I) and S1 nuclease, restriction endonuclease  Restriction endonuclease Term-Leaderberg and Messelson (1964) They are the molecular scissors, cutting down the DNA into small fragments It was found in bacterial cells, where they functioned as protective mechanism ie. Restriction-modification system(it hydrolyses the exogenous DNA which appeared in the cell through their modification-methylase They are the group of enzymes that recognize specific nucleotide sequence (4-6 base pairs) and cut only at this specific site Different endonuclease in different organism recognize different nucleotide sequences, hence have different cleavage site sometimes it cleaves both strand at opposite points and yielding blunt ended fragments. In other cases, instead of the opposite end they make a staggered cutting and forms sticky ends  Sometimes they recognize the palindromic sequence. Restriction-modification enzymes are divided into 2 classes- Class I- Type II enzymes here restriction and modification were performed by different enzymes and both the enzyme bind to the same target site Class II-type I and type II, here both these functions are performed by different subunits of same enzymes Type I are complex endonuclease having 15bp recognition sequences, cleave DNA about 1000bp away from 5’ end of sequence TCA, located in recognition site. Eg:-Eco K and Eco B  Type II cut only at one strand, 24-26bp downstream from 3’ end First type II is Eco R1 (1970) More than 3000 type II having 200 different recognition sequence Type II recognize specific sites and cleave at these sites hence they are used in mapping and reconstructing DNA in vitro  Nomenclature Named with 3 letter code in italics-Eco First letter- name of genus in capital Second and third- species Then strain number-if a strain has more than one restriction enzyme then represented as I, II, III General endonuclease-R added (Eco R I-enzyme derived from Escherichia coli carrying antibiotic resistance plasmid RI) The common recognition sequence is 4, 5 or 6 bp in length. Expected frequency can be calculated as 4ⁿ, n=length of recognition sequence  Modification of cut ends 3’ end of DNA has free OH group and 5’ end with phosphate group, for the manipulation the following modification are occur 1. Removal of 5’phosphate of vector DNA to prevent circularization of vector DNA during DNA insert integration by alkaline phosphatase 2. Addition of aphosphate group to free 5’hydroxyl group by T4 polynucleotide kinase 3. removal of 5’ and 3’ protruding ends by S1 nuclease 4. Filling in protruding ends with Klenow fragment of DNA pol1. (3 and 4 generate blunt ends, that ligated by T4 polynucleotide ligase) 5. Removal of nucleotides from 5’ end by lambda exonuclease 6. Removal of 3’ end nucleotides by exonuclease 111. (5 and 6 convert blunt end to protruding single stranded end) 7. Treatment of double stranded DNA with exonuclease Bal31 that digest both stands simultaneously from ends of DNA molecule and produces short ends of DNA molecule with blunt ends 8. Synthesis of single stranded tail at 3’ end by terminal deoaxy nucleotidyl transferase-tailing form protruding ends (poly A on DNA insert and poly T on vector DNA) and causes base pair under annealing condition 9. Linker (short self complementary double stranded oligonucleotides) molecules joined to the cut ends DNA ligase (Polynucleotide ligases) A joining enzymes used to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination. In recombinant DNA technology it is used to seal discontinuities in the sugar phosphate chains that arise when rDNA is made by joining DNA molecules from different sources. So it is considered as molecular glue, used to stick pieces of DNA together. Two enzymes are extensively used for covalently joining restriction fragments, they are E.coli DNA ligase and that encoded by T4 phage called T4 DNA ligase.  The main source of DNA ligase is T4 phage, hence the enzyme is known as T4 DNA ligase. T4 DNA ligase, enzyme is most efficient for sealing gaps in fragments with cohesive ends. They will also join blunt ended DNA molecules together. E. coli DNA ligase use (NAD) as a cofactor, while T4 DNA ligase requires ATP. In both case cofactor breaks into AMP which in turn adenylate the enzyme (E) to form enzyme AMP complex (EAC), that binds to nick containing 3' OH and 5’ PO4, ends on a double stranded DNA molecule. The 5' phosphoryl terminus of the nick is adenylated by the EAC with 3' OH terminus resulting in the formation of phosphodiester bond and liberation of AMP. After formation of phosphodiester bond, nick is sealed). DNA polymerase Polymerase enzymes synthesize copies of nucleic acid molecules by joining together nucleotides whose bases are complementary to the template strand base. The synthesis proceeds in a 5' 3' direction, as each subsequent nucleotide addition requires a free 3' OH group for the formation of the phosphodiester bond. This requirement also means that a short double stranded region with an exposed 3' OH (a primer) is necessary for synthesis to begin.  A DNA dependent DNA polymerase copies DNA into DNA, an RNA dependent DNA polymerase copies RNA into DNA. The DNA polymerase discovered by Konberg and coworkers in E.coli in 1956 is now known as DNA polymerase I, the other prokaryotic enzymes are DNA polymerase II, III, IV and V. In eukaryotes about ten such DNA polymerases have been identified. DNA polymerase operates according to three strict rules. ✓ It can copy only DNA that is unwound and maintained in the single stranded state. ✓ It adds nucleotides only to the end of an existing chain (that is it cannot establish the first link of the chain). ✓ 3. It functions in only one - the 5' to 3' direction.  The enzyme DNA polymerase 1 has, 5’3’polymerase function, 5' 3' and 3' 5' exonuclease activities. The enzyme catalyses a strand replacement reaction, where the 5' 3' exonuclease function degrades the non template strand as the polymerase synthesizes the new copy, this process is known as nick translation. By the action of proteases, DNA polymerase I degraded into fragments. The larger fragments possess polymerase and 3' to 5' exonuclease activity but lack 5' to 3' exonuclease activity. This portion of DNA polymerase 1 is called Klenow fragment. The klenow fragment is used where a single stranded DNA molecule needs to be copied. Reverse transcriptase (RTase) This enzyme is used to synthesize the copy DNA or complementary DNA (cDNA) by using mRNA as a template. It has no associated exonuclease activity. In 1970 Mizutani, Temin and Baltimore discovered that informations can also pass back from RNA to DNA, they found that retro viruses (a virus that has an RNA genome that is copied into DNA during the infection) contain RNA dependant DNA polymerase which also called as reverse transcriptase.  Cloning vectors Are cloning vehicles-used for the transport of foreign DNA into host cell Are extra chromosomal small genome like plasmid, phage, virus It is a DNA molecule that has the ability to replicate in a host cell and to which DNA insert is integrated for cloning It should have an origin of replication Properties of cloning vectors Should have autonomous replication power in a host cell Should be easy to isolate and purify Should be easily to introduce in the host cell Should have suitable marker gene for easy detection selection of transformed host cell In gene transfer programme, it should have the ability to integrate itself or the DNA insert into the host cell genome The recombinant vector should be identified or selected from those transformed by the vector molecule only It should have unique target sites for many restriction enzyme for the insertion of foreign DNA For the expression of DNA insert, a vector should contain promoter, operator, ribosome binding sites etc  Plasmid vectors Are self replicating, double stranded circular DNA and are extra chromosomes found in bacteria (some times in eukaryotes also-plasmids in yeast) F-plasmids-are capable of conjugation, which carry information for their own transfer from one cell to another-F+ to F- bacteria R-plasmids- are resistance to antibiotics Degradative plasmids-carry specific set of genes for utilization of unusual metabolites like toluene or salicylic acid Cryptic plasmids- without functional coding genes Size vary from less than 1 kb to more than 500kb It can introduced into bacterial cell through transformation (calcium chloride soln and shifting temp) or through electroporation (high voltage pulse) Should have origin of replication Are multicopy plasmids (10-20 copies/cell) or single copy plasmids some produce large numbers 1000/cell and used as vectors  pBR322 First artificial cloning vector (1977) from Ecoli plasmid Col EI (Boliver & Rodrigues) Contains origin of replication (Ori), 4361 bp, two antibiotic resistance genes (t ampicillin-ampʳ and tetracycline-tetʳ and unique recognition sites for 20 restriction endonucleases. Six of them are Bam H1, Eco RV, Sph1, Sal 1, Xma III and Nru1 present at tetracycline resistance coding gene part and Hind III and Cla I at promoter of tetracycline gene Pst I, Sca I and Pvu I are at ampicilline resistance part Phages:- Are bacterial infecting virus It follows either lytic or lysogenic cycle Prophage:-phage chromosomes integrated into bacterial chromosome and which multiplies inside the bacterial cells Eg:-phage λ (lambda) and M13 Λ-48502bp-with Ori, genes for head and tail proteins, enzymes for DNA replication, lysis and lysogeny and single stranded protruding cohesive ends of 12 bases 5’GGG CGGG CGA CCT 3’ 3’ CCC GCC GCT GGA 5’  It forms the COS site. Inside the bacterial cells it forms circular by pairing with this protruding site Have two Bam H1 site, that flank integration or excision region (20kb) Purified DNA is cut with Bam H1, 3 segments are formed. Left arm for-genes for production of head and tail; right arm-gene for replication and cell lysis and middle region for integration or excision process  Source DNA cut with Bam H1 or Sau 3A1 and are inserted into the region of integration or excision part by incubating with T4 DNA ligase and empty head and tail assemblies are added and the recombinant viral DNA is replicated inside the cell Multiplication of virus takes place by cell lysis and infection of nearby cell giving rise to plaques of lysed cells on a background or lawn of bacterial cells Two types of vectors:- λ replacement vectors and λ insertion vectors  λ replacement vectors: These vectors have two restriction sites which flank a region known as the stuffer fragment. This region can be replaced during cloning into this site. E.g. EMBLA 4 λ insertion vectors (vectors have a single restriction site) E.g. Lambda gt 10 and charon 16A Advantages The phage vectors are more efficient than plasmids for cloning of large DNA fragments- about 20 to 23 kb; for plasmid vector-less than 10 kb. It is easier to screen a large number of phage plaques than bacterial colonies for the identification of recombinant vectors. Plasmid vectors have to be introduced into bacterial cells by special methods, in contrast the phage vectors are directly tested on an- appropriate bacterial lawn (a continuous bacterial growth on an agar plate) where each phage particle forms a plaque (a clear bacteria free zone in the bacterial lawn). Cosmids: Hybrid of plasmid and phage (λ DNA) Contains 250bp of λ DNA with-cos site, sequences for binding and cleavge by terminase Has ori, restriction site, markers of plasmids Size 5kb and can accommodate 45 kb Once inside the host cell, the 12 base-pair cos ends, which were cleaved during invitro packaging, base pair and enable the linear DNA to circularize. Circular form is stable, and it is maintained as plasmid Plasmid part enable them to replicate with ori and help in selection by using marker gene for drug resistance) λ part helps for the packaging inside the coat and allows the forign genes to be transferred into cell by transduction Viruses: Eg:- CaMV; TMV, Gemini virus, Simian Virus 40, retrovirus, Ti plsamid etc It can directly insert gene by infecting host cell Some vectors used as expression vectors, since it contain powerful promoters Has high level of replication power and high level of gene expression Some are integrating their genome with host chromosome and causes the propagation of viral genome throughout the cell line YAC-Yeast artificial chromosome YAC's are linear DNA segments that contain all the molecular components required for replication in yeast, several thousand base pairs DNA, about 1Mb can be cloned. YACs are linear plasmid vectors that behave like a yeast chromosome. Each YAC occurs in two forms. The circular form in bacteria and linear form, multiplies in yeast cells. A typical YAC consists of contromere element (CEN) for chromosomal segregation during cell division, telomere(T) and origin of replication (ori) from bacteria and ARS (autonomously replicating sequence) of yeast origin, SUP4, a selectable marker  For cloning purpose, YAC is digested with restriction enzymes Bam Hl and Eco R1, the left arm and right arm become linear, with the end sequences forming the telomeres. Foreign DNA is cleaved with EcoR1 and the YAC arms and foreign DNA are ligated and then transferred into yeast host cells. The cells that contain YACs can be identified by red/ white colour selection. Non transformed yeast contain white colonies. Red colonies of yeast contain recombinant YAC molecules. e.g., YAC 3.  Other artificial chromosome vectors are- Bacterial artificial chromosome(BAC) for cloning in bacteria, Mammalian artificial chromosome (MAC) and Human artificial chromosome (HAC)-for mammalian and human cells. Bacterial artificial chromosome(BAC) BACs are DNA constructs, derived from a specific type of plasmid found in bacteria, that are used to clone large fragments of DNA in bacterial cells. It has ✓ F-factor origin of replication, which allows the plasmid to replicate within the bacterial host; ✓ selectable marker gene, usually an antibiotic resistance gene ✓ multiple cloning site (MCS), a region containing various restriction enzyme sites where foreign DNA can be inserted ✓ partitioning genes (parA and parB), which help in the stable maintenance of the plasmid during cell division  BACs can carry large DNA fragments, typically between 100,000 and 300,000 base pairs. This capacity making BACs ideal for cloning large genomic DNA fragments. BACs are often used to create genomic libraries, which are collections of DNA fragments that represent the entire genome of an organism.  Construction of rDNA Generation of DNA fragments or isolation of desired genes Cutting DNA with restriction endonucleases at precise location Selecting of small molecules of DNA capable of self replication (cloning vectors)/isolation of vectors Joining of two DNA by using DNA ligase and formation of chimeric DNA or rDNA Transfer of recombinant vectors into bacterial cell either by transformation or by using viruses Selecting of host cell with rDNA Generation of DNA fragments DNA is fragmented by using restriction enzymes or by mechanical shearing of genome or it is isolated from genomic libraries This fragments are separated by gel electrophoresis From the separated bands the required fragments are identified by using molecular probes(radioactively labelled DNA of known nucleotide sequence and are homologues to specific DNA) Suitable DNA is isolated by southern blotting techniques  Another method is to make a copy of genes from mRNA using reverse transcriptase or by artificial synthesis Place the gene in a vector Joining of broken ends of vector DNA with desired DNA is known as gene splicing. This can be done by Cohesive end ligation Most common method Restriction endonuclease (Eco R1) makes staggered cut at palindrome sequence and produce single stranded sticky ends Makes same cut in desired DNA Two will anneal and producing rDNA. Advantage of this method is Regenerating two restriction sites in the chimeric DNA, so that the foreign DNA part can be retrieved rather easily from the cloned copies of chimeric DNA by cleavage again with the same enzyme. Disadvantages are (1) The cleaved ends of a vector or of a foreign DNA may join end to end before getting inserted. (2) The recognition site particularly in the sequence to be cloned may not lie at a convenient position so that sometimes only a part of the desired segment will be inserted.  Homopolymer tailing Cut DNA at desired position both in vector and in clone Use precursor dATP poly dA at 3’ end of vector and dTTP poly dT at clone with enzyme terminal transferase Joined by annealing poly dA with poly dT by using ligase No reannealing of same DNA and very difficult to retrieve inserted DNA due to loss of recognition sites Blunt end ligation by T4 DNA ligase Here restriction enzyme is used to cut a duplex DNA at the same place in both the DNA strands. The broken ends are then used for joining with the two ends of another DNA molecule irrespective of the sequences present at the broken ends of the two DNA molecules. The T4 DNA ligase is used for this joining reaction. The disadvantage of this method is that any two broken ends may join including those belonging to the same DNA molecule.  Linkers Linkers are chemically synthesized double stranded oligonucleotides They are linked to blunt ends of vector DNA or of an insert and create a Ecol R1 site, hence the inserted DNA can be retrieved by cleavage with Ecol R1 and generate a sticky ends ADAPTORS Adaptors are, in linkers with a cohesive end. Adaptor is a short, double stranded, synthetic oligodeoxyribonucleotid , with more than one restriction site, a blunt end and a protruding cohesive end The cohesive end has a single strand extension which can base pair with a corresponding complementary cohesive end of a DNA molecule, created by a specific restriction endonuclease. After the blunt-end ligation of a target DNA, the construct can be integrated to a vector, using the cohesive end of the adaptor. Adaptors are commonly used (i) to join a bunt-ended molecule to a molecule with cohesive ends (ii) to join restriction fragments to those vectors with incompatible cohesive ends and (iii) to insert new restriction sites into cloning vectors.  Introducing of vector DNA into host cell By transformation-adding new DNA to a bacterial cell By transfection-infecting bacterial cells by using phage particles In transformation the recombinant vector is added to a flask containing a culture of E.coli. Calcium chloride is added to the flask followed by a brief heat shock. Which making appearance of holes in the cell surface membranes of E.coli making them permeable to DNA and allowing the plasmids to enter. The insert contains a selectable marker which allows for identification of recombinant molecule In transfection-Vectors that have cos sequences of lambda phage (cosmids or lambda phage vectors) are packaged in vitro into empty phage heads. The packaging produces complete lambda particles with recombinant DNA. These phage particles used to infect E.coli  Transformation and detection of recombinant molecule 1. Direct selection method Selectable marker like antibiotic resistance, color changes or any other method is used to distinguish a transformed host from an untransformed host An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant. In this technique we can not say whether the recombinants growing on such medium contain recircularized plasmid vector or recombinant plasmid plus foreign DNA fragment. Because ampr gene is present in both the recombinants. So a modified technique called insertional selection inactivation)  e.g., When the vector possess two antibiotic resistance genes, as in the case of pBR 322, i.e ampr and Tetr If the target DNA is inserted into Tetr gene, the property of resistance to tetracycline will be lost This recombinants are grown onto medium containing tetracycline, they will not grow because their Tetr gene has been inactivated. But they are resistant to ampicillin because ampr gene is functional. if the self ligated recombinants will show resistance to tetracycline and ampicillin. Therefore, they will grow on medium containing both the antibiotics and are discarded 2. Blue white selection method X gal compound (5 bromo 4 chloro 3 indolyl beta D galactoside/galactopyranoside), a colorless substrate for beta galactosidase when lactose is available, The enzyme galactosidase is synthesised The induction can also occur if a lactose analogue such as IPTG (Iso Propyl Thio Galactoside) is used. This has the advantage of being an inducer without being a Substrate for beta galactosidase. On cleavage of X gal, a blue coloured product is formed. Thus the expression of the Lac Z gene can be detected easily. This can be used either as screening method for cells or plaques, or as a system for tissue specific gene  an intact beta galactosidase gene (Lac Z) may be present in the vector and Host cells that are Lac negative are used for propagation of the phage, the lac positive phenotype, will only arise when the vector is present.  the foreign gene is inserted at a restriction site in the Lac Z gene and the vectors are grown on a medium containing X gal (ẞ galactosidase can breakdown X-gal to a blue compound), those colonies which lack the foreign DNA will appear blue because of the presence of a functional Lac Z gene that produces beta galactosidase and conversion of X gal is possible. Bacteria which form colourless colonies are the ones containing the foreign DNA and can be isolated for further cloning. When X gal is cleaved, beta galactosidase yields galactose and 5 bromo 4 chloro 3 hydroxy idole, the latter then spontaneously dimerizes and is oxidized into 5,5' dibromo 4,4' dichloro indigo, an intensely blue product, which is insoluble. X gal itself is colourless.). Colony hybridization Developed by Grustein and Hogness (1975). This technique is used to identify those bacterial colonies with 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 hybridization experiment. The procedure is as follows 1. The bacterial cells subjected to transformation are plated onto a suitable agar plate, this is the master plate. 2. The colonies on the master plate are replica plated onto a nitrocellulose filter membrane placed on agar medium. For replica plating, a block of wood or cork, of suitable diameter for the master plate, is covered with velvet cloth. This block is sterilized and then 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 the bacterial cells sticking on to 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 on the replica plate to facilitate later comparisons 3. After the colonies appear, the filter is removed from the agar plate and put on blotting paper soaked with 0.5 N NaOH solution. The alkali diffuses into nitrocellulose, lyses the bacterial cells and denatures their DNA. Thereafter the, the filter disc is nutralised by tris (hdroxymethyl) aminomethane HCL buffer by keeping high salt concentrations. 4. The filter is treated with protinase K to digest and remove the proteins. The denatured DNA remains bound to the filter 5. The filter is now baked at 80° C. to fix 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. 6. The filter is now hybridized with the radioactive probe. The probe represents the sequence of DNA segment used for transformation, generally probe DNA is labeled with P32 or 1 125 The un hybridized probe is removed by repeated washing. The colonies whose DNA hybridizes with the probe, are detected by autoradiography, only these colonies show up in the autoradiograph. 4. Plaque hybridization Developed by Benton and Davis (1977). This technique is used for screening of bacteriophages from plaque forming units. In this method lawn of E.coli cells are prepared on-agar medium which is allowed to get infected by recombinant phage particles. They infect E.coli, multiply inside cells and lyse them forming plaques. As in colony hybridization replica plate is prepared from master plate containing plaques using nitrocellulose filter, and follow the steps exactly like in colony hybridization. Recombinant phage particles isolated from the plaques are used for further study.  Some lambda vectors retain the lysogenic function, controlled by the cl gene e.g. Lambda gt 10 In such vectors, the DNA insert may be placed within the lysis repressor gene cl,so that the vector becomes cl-. As a result, cells infected by the recombinant vector give rise to clear plaques by lysis of bacterial cells, and those infected by the unaltered vector will yield cloudy or turbid plaques. Thus the recombinant vectors are readily identified and isolated. Immunological methods Here instead of radiolabelling of DNA molecules (probe), antibodies (immunoglobulins) are used to identify the colonies or plaques developed on master plates that synthesize antigens encoded by the foreign DNA present in vector (plasmid or phage). here an expression vector is designed where the foreign DNA is transcribed and translated within the bacterial cells For this the antibody specific to the concerned gene product (protein) is spread uniformly on a solid support, e.g. plastic or paper disc which is placed in contact with an agar layer containing lysed bacterial colonies or phage plaques. If any clone is producing the protein, it will bind to the antibody molecules present on the disc.  The disc is removed from the agar, is treated with a second radiolabelled (generally I125) antibody which is also specific to the same protein but in a region different from that recognized by the first antibody. The antibodies that do not react are washed off and position of antigen antibody complex is determined by autoradiography. The colonies/ plaques producing the protein are then identified and isolated from the master plate. Multiplication, expression and integration of the DNA insert in host genome Successfully transformed bacteria will carry either recombinant or non recombinant plasmid DNA. Multiplication of the plasmid DNA occurs within each transformed bacterium. A single bacterial cell placed on a solid surface (agar plate) containing nutrients can multiply to form a visible colony made of millions of identical cells. As the host cell divides, the plasmid vectors are passed on to progeny, where they continue to replicate. Numerous cell divisions of a single transfomed bacteria result in a clone of cells (visible as a bacterial colony) from a single parental cell. This step is where "cloning" got its name. The cloned DNA can then be isolated from the clone of bacterial cells. Transfer of desired gene to target cell in eukaryotes By somatic cell hybridization: by fusing of two genetically different somatic cell and form hybrid Recombinant virus mediated gene transfer DNA mediated gene transfer By direct microinjection