RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATION

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RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATION: 

RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATION MRITUNJAY KUMAR M.PHARM,2 ND SEM DEPT.OF PHARMACEUTICAL SCIENCES DIBRUGARH UNIVERSITY

INTRODUCTION: 

INTRODUCTION Definition of recombinant DNA technology:- A series of procedures used to recombine DNA segments under certain conditions, a recombinant DNA molecule can enter a cell and replicate. Organism DNA+Foreign DNA=Production of recombinant DNA,such organism are known as Transgenic organism. Deptt.of Pharmaceutical sciences,Dibrugarh University 2

History of recombinant DNA technology: 

History of recombinant DNA technology Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973. Deptt.of Pharmaceutical sciences,Dibrugarh University 3

Basic principle of recombinant DNA technology: 

Basic principle of recombinant DNA technology The DNA is inserted into another DNA molecule called ‘vector’. The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced . Deptt.of Pharmaceutical sciences,Dibrugarh University 4

Basic principle of recombinant DNA technology: 

Basic principle of recombinant DNA technology Deptt.of Pharmaceutical sciences,Dibrugarh University 5

Gene cloning: 

Gene cloning It can be defined as the isolation and amplification of an individual gene sequence by insertion of that individual gene sequence into a bacterium where it can be replicated. Deptt.of Pharmaceutical sciences,Dibrugarh University 6

There are some steps involved in gene cloning:- 1.Isolation of gene of interest 2.A fragment of DNA to be cloned is incorporated into a small replicating DNA molecule called a vector. 3.The recombinant vector is introduced into a host cell by transformation. 4.Cell that contains rDNA are selected. 5.Growth and multiplication of rDNA . Deptt.of Pharmaceutical sciences,Dibrugarh University 7

Isolation of the gene of interest:-: 

Isolation of the gene of interest:- 1.Enzyme for cutting and joining the DNA 2.Cloning vehicle or Vectors 3.DNA fragments or gene liberaries 4.Selection of a clone of transformed cells that has acquired the rDNA molecules. Deptt.of Pharmaceutical sciences,Dibrugarh University 8

Enzyme for cutting and joining the dna: 

Enzyme for cutting and joining the dna Cutting DNA molecule at the specific sites with restriction endonucleases . There are three types of restriction endonuclease :- TYPE I:- they are complex nucleases. TYPE II:- they are simple enzymes that consist of a single polypeptide. TYPEIII:- they contain two different subunit and require ATP and Mg++ as cofactor. Deptt.of Pharmaceutical sciences,Dibrugarh University 9

Joining of dna molecule: 

Joining of dna molecule The enzyme used to join DNA fragments are called DNA ligase. This is the final step in construction of a recombinant DNA molecule,process is called Ligation. Deptt.of Pharmaceutical sciences,Dibrugarh University 10

vectors: 

vectors A vector should have the following features:- 1) It must have a replicon 2) It should have several marker gene 3) It should have a unique cleavage site within one of the marker gene 4) It should replicate independently Example of vectors used in rDNA technology:- plasmid,cosmids,bacteriophage,Ti -plasmid Deptt.of Pharmaceutical sciences,Dibrugarh University 11

plasmid: 

plasmid They are double stranded closed circular DNA molecules which exist in the cell as extra chromosomal unit. They are self replicating. They replicate and inherited independently. There are 3 general classes of plasmids i ) Virulence plasmid ii) Drug resistance plasmid iii) plasmid which encode genes required for bacterial conjugation. Deptt.of Pharmaceutical sciences,Dibrugarh University 12

PowerPoint Presentation: 

Plasmid range in size 1 to 200kb. Plasmid can be single copy plasmid that are maintained as one plasmid DNA per cell. Multi copy plasmid that are maintained as 10-20 genome per cell. Most plasmids used in cloning have a relaxed mode of replication system. Plasmid vector used for cloning is specially developed by adding certain features:- 1. Reduction in size of plasmid to a minimum to expand the capacity of vector to clone large fragments. 2. It should contain origin of replication. 3. introduction of selectable marker genes. Deptt.of Pharmaceutical sciences,Dibrugarh University 13

cosmids: 

cosmids Cosmids are plasmid vectors that contain a bacteriophage lambda cos site,which directs insertion of DNA into phage particles. The development of Cosmids vectors was based on the observation that a 200 bp DNA sequence in the lambda genome called cos is required for DNA packaging into the phage particle during lytic infection. Cosmids were developed to overcome the technical problem of introducing large pieces of DNA into E.Coli . Deptt.of Pharmaceutical sciences,Dibrugarh University 14

PowerPoint Presentation: 

Cosmids cloning vectors with DNA inserts of 30 to 45 Kb can be packaged in vitro into lambda phage particles. Cosmids vectors posses an origin of replication, a selectable genetic marker (antibiotic resistance),and suitable sites. Cosmids are ideal vector for genome mapping. It can multiply in large copy number like plasmid vector. ADVANTAGE:- large size of insert DNA can be cloned & DNA can be introduced into the host using bacteriophage derived by in vitro packaging. DISADVANTAGES:- It is difficult to store bacterial host as glycerol stock & in vitro packaging is needed to maintain Cosmids inside the viral heads. Deptt.of Pharmaceutical sciences,Dibrugarh University 15

Bacteriophage vectors: 

Bacteriophage vectors Bacteriophage are viruses that infect bacteria. These are usually called phages and it is constructed from two basic components: protein and nucleic acid genome. Phages can be double stranded (T2,T4,T6,lambda) or single stranded(phage 174,M13). There are both DNA(T2,T4,T6) and RNA(MS2) phages. Phages contain an origin of replication. The process is known as transduction by which phages enter into the bacteria. Deptt.of Pharmaceutical sciences,Dibrugarh University 16

Ti-plasmid: 

Ti-plasmid Most cloning vectors for plants are based on the Ti plasmid,which is not a natural plant plasmid but belongs to a soil bacterium Agrobacterium tumefaciences. This bacterium invades plants tissue,causes a cancerous growth called a crown gall. During infection,a part of Ti plasmid called the T-DNA is integrated to the plant chromosomal DNA. There are Ri plasmid present in A . rhizogenes which causes hairy root disease in plants. Deptt.of Pharmaceutical sciences,Dibrugarh University 17

Contd…..: 

Contd ….. The Ti plasmid are large circular DNA molecule,upto 200 Kb in length. They are independent replicating unit present in bacteria. Ti plasmid have major regions of virulence, origin of replication,conjugation and catabolism of opines. Deptt.of Pharmaceutical sciences,Dibrugarh University 18

Marker gene: 

Marker gene Monitoring and detection of plant transformation systems in order to know whether the DNA has been successfully transferred into recipient cell is done with the help of set of genes. The marker gene is introduced into the plasmid along with the target gene. They are of two types : Reporter gene & Selectable marker gene Deptt.of Pharmaceutical sciences,Dibrugarh University 19

Gene transfer method: 

Gene transfer method Various gene transfer techniques are grouped in two main categories: 1) Vector mediated gene transfer(bacteria and virus mediated) 2) Vectorless or Direct DNA transfer 1) Vector mediated gene transfer :-The term plant gene vector applies to potential vectors both for transfer of genetic information between plant and the transfer of genetic information from other organism( bacteria,fungi and animal) to plant. Deptt.of Pharmaceutical sciences,Dibrugarh University 20

Agrobacterium mediated transformation: 

Agrobacterium mediated transformation Agrobacterium is commonly used for gene transfer. A.Tumefaciences: induces crown gall disease A.rhizogenes: induces hairy root disease A.radiobacter: It is an avirulent strain. Transformation technique using Agrobacterium Agrobacterium mediated plant transformation techniques are now extensively used to generate transgenic plants. Deptt.of Pharmaceutical sciences,Dibrugarh University 21

Requirement for agrobacterium mediated gene transfer: 

Requirement for agrobacterium mediated gene transfer The plant explants must produce acetosyringone or other related compounds for activation of vir genes or Agrobacterium may be preinduced with synthetic acetosyringone. The induced agrobacteria should have acess to cells that competent for transformation. Transformation competent cells should be able to regenerated into whole plants. Deptt.of Pharmaceutical sciences,Dibrugarh University 22

3 approaches for inoculation of agrobacterium: 

3 approaches for inoculation of agrobacterium Infection of wounded plants Co-cultivation Leaf disc method VIRUS MEDIATED GENE TRANSFER Ex : Caulimovirus group , cauliflower mosaic virus( CaMV ) potential vector for introducing foreign gene into the plants. Gemini virus( curly top virus and maize streak virus):The potential of gemini viruses as gene cloning vector for plants stems from work on several plant diseases now recognized by these agents. Deptt.of Pharmaceutical sciences,Dibrugarh University 23

Direct dna transfer: 

Direct dna transfer Direct gene transfer has proved to be a simple and effective technique for the introduction of foreign DNA into plant genome. It has been further sub-divided into three categories:- 1) Physical gene transfer method 2) Chemical gene transfer method 3) DNA imbibition by cell,tissue and organs Deptt.of Pharmaceutical sciences,Dibrugarh University 24

Application of recombinant dna technolgy: 

Application of recombinant dna technolgy Recombinant bacteria in Industry Recombinant bacteria in Medicine Recombinant bacteria in Agriculture Production of Transgenic animal Production of Transgenic plants Bioprocessing Deptt.of Pharmaceutical sciences,Dibrugarh University 25

Bibliography:-: 

Bibliography:- Chawla H.S”Introduction to plant biotechnology”3 rd Edition,Oxford & IBH Publishing Co.Pvt.Ltd,New Delhi,page no:213 to 250. http://www.ied.edu.hk/biotech/eng/classrm/explain/gene5.htm www.wikipedia .com Deptt.of Pharmaceutical sciences,Dibrugarh University 26

Deptt.of Pharmaceutical sciences,Dibrugarh University 27