DNA Recombinant technology and uses .ppt

Molecular Techniques

1. MR. R. SUSIL LECTURER GANGA INSTITUTE OF HEALTH SCIENCES MEDICAL LABORATOTECHNOLOGY

2. DNA RECOMBINANT TECHNOLOGY

3. Recombinant DNA technology is the joining together of DNA molecules from two different species. The recombined DNA molecule is inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. T he focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Recombinant DNA technology is based primarily on two other technologies, cloning and DNA sequencing. Once a segment of DNA has been cloned, its nucleotide sequence can be determined. Knowledge of the sequence of a DNA segment has many uses. What is recombinant DNA technology?

4. DNA RECOMBINANT TECHNOLOGY

5. Clone is a group of individual cells or organisms descended from one progenitor. The members of a clone are genetically identical, because cell replication produces identical daughter cells each time. DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. DNA cloning

6. DNA cloning

7. It is used in the production of hormones, vitamins and antibiotics. Gene cloning finds its applications in the agricultural field. Nitrogen fixation is carried out by cyanobacteria wherein desired genes can be used to enhance the productivity of crops and improvement of health. Identifying and detecting a clone containing a particular gene which can be manipulated by growing in a controlled environment It is used in gene therapy where a faulty gene is replaced by the insertion of a healthy gene. Medical ailments such as leukaemia and sickle cell anaemia can be treated with this principle. Applications Of Gene Cloning

8. Step-1. Isolation of Genetic Material. The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form free from other macromolecules. Step-2.Cutting the gene at the recognition sites. The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’. Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR). It is a process to amplify a single copy of DNA into thousands to millions of copies once the proper gene of interest has been cut using restriction enzymes. Process of Recombinant DNA Technology

9. Process of Recombinant DNA Technology Step-4. Ligation of DNA Molecules. In this step of Ligation, the joining of the two pieces a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase. Step-5. Insertion of Recombinant DNA Into Host. The recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in the form of the manufactured protein under optimal conditions. Process of Recombinant DNA Technology

10. Process of Recombinant DNA Technology

11. DNA is extracted from the organism under study and is cut into small fragments of a size suitable for cloning. Cleaving the DNA with a restriction enzyme. Restriction enzymes are extracted from several different species and strains of bacteria, in which they act as defense mechanisms against viruses. They can be thought of as “ molecular scissors, ” cutting the DNA at specific target sequences. The donor DNA and the vector DNA are both cut with the same enzyme. The resulting molecule is called recombinant DNA. It is recombinant in the sense that it is composed of DNA from two different sources. Creating the clone

12. The original mixture of transformed bacterial cells is spread out on the surface of a growth medium in a flat dish (Petri dish) so that the cells are separated from one another. These individual cells are invisible to the naked eye, but as each cell undergoes successive rounds of cell division, visible colonies form. Each colony is a cell clone, but it is also a DNA clone because the recombinant vector has now been amplified by replication during every round of cell division. The Petri dish, which may contain many hundreds of distinct colonies, represents a large number of clones of different DNA fragments. This collection of clones is called a DNA library. Creating the clone

13. Creating the clone

14. Creation of a cDNA library begins with messenger ribonucleic acid (mRNA) instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA. The resulting DNA molecules are called complementary DNA (cDNA). A cDNA library represents a sampling of the transcribed genes, whereas a genomic library includes untranscribed regions. Both genomic and cDNA libraries are made without regard to obtaining functional cloned donor fragments. cDNA library

15. cDNA library

16. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning. Plasmid

17. Plasmid

18. Plasmids replicate independently within a cell. They must possess a stretch of DNA that can act as an origin of replication. The self-replicating unit the plasmid, is called a replicon. A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep) Repeating units called iterons, DnaA boxes, and an adjacent AT-rich region. Smaller plasmids make use of the host replicative enzymes to make copies of themselves. Larger plasmids may carry genes specific for the replication of those plasmids. A few types of plasmids can also insert into the host chromosome, and these integrative plasmids are sometimes referred to as episomes. Properties and characteristics of plasmid

19. Properties and characteristics of plasmid

20. Fertility F-plasmids , which contain tra genes. They are capable of conjugation and result in the expression of sex pili. Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or antibacterial agents and also known as R-factors. Col plasmids , which contain genes that code for bacteriocins, proteins that can kill other bacteria. Degradative plasmids , which enable the digestion of unusual substances, e.g. toluene and salicylic acid. Virulence plasmids , which turn the bacterium into a pathogen. e.g. Ti plasmid in Agrobacterium tumefaciens Classifications and types of plasmids

21. Classifications and types of plasmids

22. Plasmid

23. Vector

24. Restriction Enzyme Genome

25. Bacteria – Single-celled microbes that were one of the first types of lifeforms to evolve on Earth; they can exist independently or inside other organisms. Episome – In bacteria, a plasmid that can be inserted into the chromosome. In eukaryotes, plasmid refers to non-chromosomal DNA that can be replicated in the nucleus, such as a virus.

26. Conjugative – A category of plasmids that start the process of sexual conjugation in bacteria. Bacteriocin – a protein produced by a plasmid in a bacterium that kills other bacteria of a closely related strain.

27. DNA technology is also used to detect the presence of HIV in a person. 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 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. Recombinant DNA technology is used for the production of Insulin. Application of Recombinant DNA Technology