RECOMBINANT DNA (RDNA) TECHNOLOGY

                   BY- ABHISHEKA G.(MSIWM013)

INTRODUCTION:

1.Recombinant DNA or RDNA technology is defined as the procedure of joining DNA molecules of two different species together and inserted into the host organism to produce a variety of new genetic combinations. This is also known as Genetic engineering.

2. The DNA fragments are selected from two different species and combined. This technique was developed by two scientists namely Boyer and Cohen in 1973.

3. The DNA molecule which is inserted into another DNA molecule is called a VECTOR. The recombinant vector is then introduced into a host cell where it replicates itself, and the new gene is produced. This is the basic principle behind Recombinant DNA technology.

TOOLS OF THE RECOMBINANT DNA TECHNOLOGY:

  1. Restriction endonucleases: These are used to cut DNA molecules at specific sequences into many smaller DNA fragments.
  2. Plasmids: These are extrachromosomal circular DNA present in the bacteria, which can replicate independently. During cloning, these plasmids carry drug resistance genes that are used for selection. Foreign DNA can be placed into a plasmid and it is replicated further.
  3. DNA ligase: This enzyme is used to join the two pieces of DNA together.
  4. Foreign DNA: This is also known as passenger DNA, which contains desired gene sequences.
  5. Vector: It is a vehicle used to insert the desired DNA into the host cell. Some of the vectors used are Plasmid DNA, Bacteriophage DNA, Yeast DNA, Viral DNA, Bacterial DNA, etc.

GOALS OF RDNA TECHNOLOGY:

  1. To isolate and characterize a gene or DNA from an organism.
  2. To eliminate undesirable phenotypic characters.
  3. To combine the needy and beneficial traits of two or more organisms.
  4. To make desired alterations in one or more isolated genes or DNA
  5. Inserting the altered genes or DNA into the host cell of another organism.
  6. To synthesize new genes using artificial methods.
  7. To alter the genome of the organism
  8. Understanding the diseases which transmit due to heredity.
  9. Understanding the treatment for heredity related disorders.
  10. To create new gene combinations.

PROCEDURE TO PREPARE RDNA:

1 Isolation of DNA from the organism: The cells are lysed using detergent mixtures, which creates pores in the plasma membrane. Then the mixture of cell contents is treated with protease and RNAase enzymes. The enzyme protease destroys the proteins present in the mixture and the enzyme RNAase destroys the RNA molecules present in the mixture. Then the mixture is centrifuged and the supernatant containing the DNA is transferred into a clean test tube and the DNA precipitated with the addition of ethanol.

2. Insertion of foreign genes into vectors: By using plasmid as a vector, isolated from the bacterial cell and treated with restriction enzymes and target DNA is obtained and it is placed into a vector to produce recombinant DNA.

3. Insertion of Recombinant DNA into host cell: The plasmid containing the foreign DNA is placed into a bacterial or host cell for multiplication.

4.Transformation: The vector is used as a vehicle to transport the gene to host cell, bacterium or other living cells are used as vectors. The vector is multiplied in the host cell and produces many identical copies, which are similar to both DNA and gene present in the DNA.

5. Cloning: After the division of the host cell the rDNA copies produced are transmitted to the progeny and further vector replication takes place in the progeny cell, with the continuous division of cells, a clone of identical host cells is formed. Each clone contains one or more copies of the rDNA molecule. Later the identical host cells are lysis and rDNA molecules are separated from the host cells.

APPLICATIONS OF RDNA TECHNOLOGY:

  1. This technology helps to grow crops which are resistant diseases and pesticides, crops of our choice, fruits, and flowers of attractive colors.
  2. This technique is employed in the production of artificial insulin and to deliver the drugs to target sites.
  3. Used in Molecular diagnosis of diseases.
  4. Used in Gene therapy.
  5. Employed in DNA fingerprinting.
  6. Used in the production of vaccines and pharmaceutical products.
  7. In the production of monoclonal antibodies.

PLASMID AND TYPES OF PLASMIDS

BY: RAHUL ANDHARIA (MSIWM001)

Introduction:

  • The plasmids are defined as naturally occurring, stable extra-chromosomal genetic elements found in all three major groups of microbes. (Archaea, bacteria and eukaryotes).
  • Among eukaryotes, plasmids are found in fungal cells and mitochondria of some plants.
  • In addition to naturally occurring plasmids, a wide range of plasmids have been engineered for specific applications in Recombinant DNA technologies and in genetic engineering.
  • Plasmids may be composed of single stranded or double stranded DNA or RNA.
  • Plasmids may be linear or circular. The size of plasmids range from 1kb to over 200kb. Plasmids replicate independently of the chromosome of cell.
  • Plasmids provide bacteria with genetic advantages, such as Antibiotic resistance, which is conferred by genes present in the plasmids.
  • The other important known function of plasmids is they contain genes that enhance survival of organism, either by killing the organism or by producing toxins against it.
  • Certain plasmids provide selective advantage to the host under specific conditions.

History:

  • The term plasmid was coined for the first time in the year 1952 by Lederberg.
  • He worked on experiments with bacterial conjugation to map the genome and revealed two groups of linked genes; (a) Main bacterial chromosomes and (b) a second chromosome that is present only in some bacterial cells.
  • This class of extra chromosomal elements is termed as Plasmids.
  • Plasmids are believed to be the evolutionary ancestors of viruses.

Structure of Plasmids:

  • Plasmids are generally composed of circular double chains of DNA. The two ends of plasmids are held together by covalent bonds.
  • Origin of Replication (ori): it refers to the site at which replication begins. In plasmids, this ori is generally composed of A-T base pairs, which are much easier to separate during replication. As plasmids are smaller in size, they have one to few origins of replication sites. Regulatory elements are also present at the ori site. For example- Rep proteins.
  • Multiple cloning sites: this is also called as polylinker. A short DNA sequence consists of few sites for cleavage by restriction enzymes. At the cleavage site, strand can be cut by different polylinkers. One main advantage of multiple cloning sites in plasmids is that it does not hinder the rest of the plasmid during the process and also possess unique restriction enzymes, which can cut the plasmid at specific points to allow DNA insertion.
  • Antibiotic Resistance gene: This is one of the main components in plasmids which help in Drug resistance. By a process of conjugation, plasmids transfer from one bacteria to the other and during this process they are capable of conferring antibiotic resistance properties to the bacteria.
  • A Promoter region: this region helps in the process of transcription and in recruitment of transcriptional machinery.
  • Primer binding site: this is specifically used for PCR amplification or for DNA sequencing and generally refers to short sequence of DNA on a single strand.

Types of Plasmids:

  1. F Plasmid (fertility factor):
  2. It is the best studied conjugative plasmid. It plays a major role in E.coli conjugation.
  3. It is about 100kb in size.
  4. It has genes responsible for cell attachment and plasmid transfer.
  5. Tra operon (has tra genes responsible for nonsexual transfer of genetic material in bacteria) is important in F plasmid.
  6. It contains 28 genes and these genes direct the formation of sex pilus (helps in transfer of DNA in bacteria during bacteria conjugation).
  7. F factor also possesses insertion sequence (short sequences which can acts as transposable elements) that assists plasmid interaction into host chromosome.
  • Ti Plasmids:
  • It is present in soil bacterium, Agro bacterium Tumifaciens.
  • It induces tumor in plants and hence commonly called as Tumor inducing plasmid.
  • It is a large plasmid. Generally, its size varies from 180-250kb.
  • It contains T-DNA region of about 23-25kb. This region is generally transferred into plant cells.
  • Ti plasmids can be of 3 types based on kind of opines (carbon compounds found primarily in crown gall tumors) they encode for; Octopine, Nopaline, Agropine.
  • Opines are neither naturally found in plants and nor required by plants. Agro bacterium uses it as a source of carbon and nitrogen for its growth and multiplication.

Mechanism of infection:

  • Formation of wound is essential in plants for the infection by Agro bacterium Tumifaciens.
  • The lipopolysaccharides present on the bacterial cell walls and Polygalacturonic acid of damaged plant cell wall helps in the process of attachment of agro bacterium to plant cell.
  • Damaged plant cell wall also produces Acetosyringone, a low molecular weight phenolic compound. This compound induces transcription of Virulence genes (Vir genes) present on Ti plasmid.
  • Enzymes produced by virulence genes, makes a nick in one strand of plasmid at two points.
  • This produces single stranded DNA fragment which is then carried to plant cells.
  • T-DNA of Ti plasmid integrates with plant cell chromosome. As a result of this plant cells produces opines. This opines helps in growth and multiplication of Ti plasmid.
  • T-DNA also codes for phytohormones like auxins and cytokinin. This hormones leads to disorganized proliferation plant cells causing tumors called Crown Gall tumors.
  • R plasmid (resistance plasmids):
  • R plasmids are well studied group of plasmids. Their role is to confer antibiotic resistance and inhibits various other growth inhibitors.
  • R plasmids have genes that encode for enzymes that are able to destroy or modify antibiotics.
  • R plasmids evolve rapidly and can easily acquire additional resistant determining genes.
  • A single plasmid transfer can turn a drug sensitive bacterium into a multiple drug resistant strain.
  • Broad host range plasmids that carry multiple antibiotic resistant genes are of great medical concern because they can be transferred to a wide range of bacterial species.
  • Degradative plasmids:
  • Degradative plasmids are Plasmids that encode genes required for the metabolism of wide range environmental contaminants.
  • As they can be transferred between microorganisms, they can provide a means for the rapid horizontal spread of degradative genes among natural microbial populations.
  • Direct seeding of plasmids by Soil bioremediation by borne genes into native soil is a potential useful way to enhance the degradation of environmental pollutants.
  • 2-4-D plasmids were found in strains isolated by enrichment on 2-4-D as the sole source of carbon and energy and some of them were found to degrade herbicide with similar structure.
  • Strain of Pseudomonas Putida called NCIB was formed to possess plasmid PDTG1 with 83,042 base pairs. This plasmid also encodes enzymes for Naphthalene degradation.
  • COL plasmids (col-colicine bacteriocines):
  • Col plasmids are present in different genes of E.coli.
  • They contain genes that control the synthesis of proteins called Colicines (proteins which has the ability to kill other bacterial strains and are often used by host bacterium).
  • This colicines inhibit growth of related bacteria that lacks Col plasmid.
  • Different types of colicines exhibit different mode of action.
  • Col-B induces damage of cytoplasmic membrane of the target bacteria.
  • Example of Col plasmids- (Col E2 and Col E3) causes degradation of nucleic acids.
  • Col plasmids are may be self transmissible or non-self transmissible (this non self transmissible may be mobilized by F plasmids).
  • This means that when F+ cell contains Col E plasmid, this plasmid can integrate with F factor and gets transported to Fcell during conjugation.