Plant Biotechnology Recombinant DNA Technology ppt.pptx

Biotechnology, recombinant DNA Technology

1. Recombinant DNA Technology

2. Introduction A technique mainly used to change the phenotype of an organism (host) when a genetically altered vector is introduced and integrated into the genome of the organism. So , basically, this process involves the introduction of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is introduced is the recombinant gene and the technique is called the recombinant DNA technology. Inserting the desired gene into the genome of the host is not as easy as it sounds. It involves the selection of the desired gene for administration into the host followed by a selection of the perfect vector with which the gene has to be integrated and recombinant DNA formed. Thus the recombinant DNA has to be introduced into the host. And at last, it has to be maintained in the host and carried forward to the offspring.

3. What is Recombinant DNA Technology ? The exchange of genetic information between DNA segments of the same species is termed genetic recombination . However, with the advancement of this technology , one can transfer genes of one species to another artificially . The technology used for producing artificial DNA through the combination of different genetic materials (DNA) from different sources is referred to as  Recombinant DNA Technology .  OR- It is the set of techniques that enable the DNA from different sources to be isolated, identified and recombinant so that the new characteristics can be introduced into an organism. Recombinant DNA technology is popularly known as genetic engineering . 

4. Goals of Recombinant DNA Technology : Isolation and characterization of genes. To make desired modification / alteration in isolated genes. To return the altered gene to living cells with new desired characters (gene cloning). Artificial synthesis of new genes and add new character. Modification / alteration of organism’s genome. Understanding of hereditary diseases and related cures. Improving of the human genome.  

5. Recombinant DNA in a living organism was first achieved in 1973 by  Herbert Boyer and  Stanley Cohen , who used  E. coli restriction enzymes to insert foreign DNA into plasmids . Paul Berg developed the 1st recombinant DNA molecules that combined DNA from SV 40 virus and lambda phage . There are multiple steps, tools and other specific procedures followed in the recombinant DNA  technology, which is used for producing artificial DNA to generate the desired product.

6. Tools of Recombinant DNA Technology The enzymes which include the restriction enzymes help to cut, the polymerases- help to synthesize and the ligases - help to bind. “ Restriction enzymes are the enzymes produced by certain bacteria that have the property of cleaving DNA molecule at or near specific base sequences.” The restriction enzymes used in recombinant DNA technology play a major role in determining the location at which the desired gene is inserted into the vector genome. They are two types, namely Endonucleases and Exonucleases. The Endonucleases cut within the DNA strand whereas the Exonucleases remove the nucleotides from the ends of the strands. The restriction endonucleases are sequence-specific which are usually palindrome sequences and cut the DNA at specific points. They scrutinize the length of DNA and make the cut at the specific site called the restriction site. This gives rise to sticky ends in the sequence. The desired genes and the vectors are cut by the same restriction enzymes to obtain the complementary sticky notes, thus making the work of the ligases easy to bind the desired gene to the vector .

7. 2. The vectors – a DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism. Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have a very high copy number. Agrobacterium tumefaciens  is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor -inducing or Ti plasmid to susceptible plant cells.  Ti -plasmid, short for tumour -inducing plasmid, is an extrachromosomal molecule of DNA found commonly in the plant pathogen Agrobacterium tumefaciens . (The Ti -plasmid in the bacteria is known to induce crown gall disease in plants by transferring crucial regions from the plasmid. These crucial regions were seen to modify the plant cells into a tumour to produce synthetic plant hormones and cause crown gall. This led the scientists to believe that there is a scope for bioengineering techniques to modify the plants using Ti -plasmid for our own use .) The vectors are made up of an origin of replication- This is a sequence of nucleotides from where the replication starts, a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted .

8. Features of Ti Plasmid Virulence Region : The virulence region codes for virulence genes that are responsible for the transfer of T-DNA to the plant cells and also recruiting various effector proteins for infecting the plant cells. T-DNA: The T-DNA region is the crucial region that gets transferred to the plant cell for infection. It is approximately 15-20 kbp in length and is transferred to the plant cell via means of genetic recombination. Opine Catabolism: The opine catabolism region is the region from where the bacteria sources its nutrients for the whole process. Opines are derivatives of amino acid or sugar phosphates that can be catabolized to use in the form of nutrients. The types of opines found in Ti -plasmid are nopaline and octopine types. Origin of Replication: The origin of replication is the region where replication of the plasmid is initiated.

9. 3. Host organism – into which the recombinant DNA is introduced. The host is the ultimate tool of recombinant DNA technology which takes in the vector engineered with the desired DNA with the help of the enzymes. There are a number of ways in which these recombinant DNAs are inserted into the host, namely – microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc.

10. Steps of Recombinant DNA Technology 1 Isolation   of DNA 2 Cutting of DNA/Restriction Enzyme Digestion 3 Amplifying of DNA 4 Joining of DNA 5 Insertion of rDNA into a Host 6 Recombinant Cell Isolation

11. Isolation  of DNA DNA is isolated in its pure form, which means they are devoid of other macromolecules. In rDNA technology, the initial step is to extract the desired DNA in its purest form, that is, free of extraneous macromolecules. Because DNA coexists with other macromolecules such as RNA, polysaccharides, proteins, and lipids within the cell membrane, it must be separated and purified using enzymes such as lysozymes, cellulase , chitinase , ribonuclease, and proteases. Other enzymes or treatments can remove other macromolecules. The DNA eventually precipitates out as fine threads as a result of the presence of ethanol. After that, the pure DNA is spooled out.

12. 2. Cutting of DNA/Restriction Enzyme Digestion For this step, the restriction enzymes are quite vital. It helps to identify the location wherein a designated gene is introduced into a vector genome. The said reaction is known as restriction enzyme digestions. They entail incubating pure DNA with a restriction enzyme of choice at conditions that are appropriate for that enzyme. The 'Agarose Gel Electrophoresis' technology displays the restriction enzyme digestion's progress, it helps in separation of large DNA fragments. This method entails passing the DNA across an agarose gel. When current is applied, negatively charged DNA flows to the positive electrode and is divided into different sizes. This permits the digested DNA fragments to be separated and snipped out. The same method is used to process the vector DNA.

13. 3. Amplifying of DNA Copies of genes are amplified through PCR or polymerase chain reaction. It is essentially a process to increase a single DNA copy into several copies after the desired gene of interest is cut with restriction enzymes.  It allows a single copy or a few copies of DNA to be amplified into thousands or millions of copies. The following components are used in PCR reactions that are conducted on 'thermal cyclers': Template : DNA that has to be amplified. Primers : oligonucleotides are tiny, chemically produced oligohnucleotides that are complementary to a DNA region. Enzyme : DNA polymerase. Nucleotides: The enzyme is required to lengthen the primers. PCR can be used to amplify the cut DNA fragments, which can subsequently be ligated with the cut vector.

14. 4. Joining DNA The vector and a section of DNA are joined in this step. It is achieved with the help of the enzyme DNA ligase .  With the same restriction enzyme, the pure DNA and the vector of interest are cut . This yields the cut DNA fragment and the cut vector, both of which are now open . Ligation is the process of putting these two parts together with the enzyme 'DNA ligase .‘ The resulting DNA molecule is a hybrid of the interest molecule and the vector DNA molecules. Recombination is the term used in genetics to describe the merging of different DNA strands . As a result, this new hybrid DNA molecule is known as a recombinant DNA molecule, and the process is known as recombinant DNA technology.

15. 5. Insertion of rDNA into a Host Here rDNA is added to the recipient host cell, and the entire process is called transformation. Post insertion, the recombinant DNA multiplies and manifests as manufactured protein under favorable conditions . The recombinant DNA is then transferred into a recipient host cell, most commonly a bacterial cell, in this stage. The term for this procedure is 'Transformation .‘ Bacterial cells have a hard time accepting foreign DNA. As a result, they are given treatments to make them 'capable' of accepting new DNA. Thermal shock, Ca++ ion therapy, electroporation, and other procedures may be applied.

16. 6. Recombinant Cell Isolation A mixed population of converted and non-transformed host cells results from the transformation process. Only the transformed host cells are filtered during the selection procedure. The marker gene of the plasmid vector is used to distinguish recombinant cells from non-recombinant cells. PBR322 plasmid vector, for example, comprises two marker genes (Ampicillin resistant gene and Tetracycline resistant gene). When pst1 RE is utilized, it eliminates the Ampicillin resistance gene from the plasmid, causing the recombinant cell to become Ampicillin sensitive.

17. Application of Recombinant DNA Technology Recombinant DNA technology is widely used in Agriculture to produce genetically-modified organisms such as   Flavr Savr tomatoes, golden rice rich in proteins, and Bt -cotton to protect the plant against ball worms and a lot more. Gene Therapy – It is used as an attempt to correct the gene defects which give rise to heredity diseases. Clinical diagnosis – ELISA is an example where the application of recombinant. In the field of medicines, Recombinant DNA technology is used for the production of Insulin . DNA technology is also used to detect the presence of HIV in a person.

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