CHAPTER 6: RECOMBINANT DNA TECHNOLOGY

YEAR III PHARM.DDR. V. CHITRA

INTRODUCTION

DNA : DNA is deoxyribose nucleic acid . It is made up ofa base consisting of sugar, phosphate and one nitrogenbase.The nitrogen bases are adenine (A) guanine (G)cytosine(C) and thymine(T). The nitrogen bases aregrouped as

PURINES : Adenine and guanine.

PYRAMIDINES : Cytosine, uracil and thymine.

The sequence of bases are in pairs like A&T and G&C and structure is double helix.

What is rDNA ?

Recombinant DNA is genetically engineered DNA prepared by transplanting or splicing genes from one species into the cells of a host organism of a different species .

Thus the name recombinant DNA is also referred to as “CHIMERA”.

PRINCIPLES

Generation of DNA fragments and selection of the desired piece of DNA.

Insertion of the selected DNA into a cloning vector(ex : plasmid) to create a cloning vector or a chimeric DNA.

Introduction of the recombinant vectors into the host cells(bacteria).

Multiplication and selection of clones containing the recombinant molecules.

Expression of the gene to produce the desired product.

RESTRICTION ENDONUCLEASES

These are the bacterial enzymes that can cut / split DNA at specific sites.Ex: E.coli R1, Hind 3.

RECOGNITION SEQUENCE : It is the site where the DNA is cut by a restriction endonuclease. Restriction endonucleases can specifically recognize DNA with a particular sequence of 4-8 nucleotides and cleave.

CLEAVAGE PATTERNS : The cut DNA fragments may have sticky ends or blunt ends.

DNA LIGASES

The cut DNA fragments are covalently joined together by DNA ligases.

They form a phosphodiester bond between the phosphate group of 5’- carbon of one deoxyribose with hydroxyl group of 3’- carbon of another deoxyribose.

Ex : DNA polymerase 1- Synthesis DNA complementary to a DNA template.

HOST CELLS

Host cells are the living systems or cells in which the carrier of recombinant DNA molecule or vector can be propagated.

Prokaryotic hosts : Escherichia coli, bacillus subtilis.

Eukaryotic hosts : Yeast, sacchromyces cerevisiae.

VECTORS

Vectors are DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self replicating in host cell.

PlasmidBacteriophagesCosmidsArtificial chromosome vectors(I) Human artificial chromosome (HAC)(II) Yeast artificial chromosome(III) Bacterial artificial chromosome(BAC)

PROCEDURES

TRANSFORMATION : The first step in transformation is to select a piece of DNA to be inserted into a vector.

The second step is to cut that piece of DNA with a restriction enzyme and then ligase the DNA insert into the vector with DNA Ligase.

The insert contains a selectable marker which allows for identification of recombinant molecules. 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.

The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA.

NON-BACTERIAL TRANSFORMATION :This is a process very similar to transformation. The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.

In microinjection, the DNA is injected directly into the nucleus of the cell being transformed.

PHAGE INTRODUCTION : Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packagings of a vector is used.

This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.

CONJUGATION : It is a natural microbial recombinant process during which two live bacteria come together join by cytoplasmic bridges and transfer single stranded DNA(from donor to recepient).

ELECTROPORATION METHOD : It is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse.Electric shocks can also induce cellular uptake of exogenous DNA from the suspending solution.

LIPOSOME MEDIATED GENE TRANSFER(LIPOFECTION)On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside liposomes. These liposomes can adhere to cell membranes and fuse with them to transfer DNA fragments.

GENE CLONING

GENE CLONING : The technique of making many copies of a gene.Generally genomic DNA is ideal for cloning, but the DNA contains non coding sequences(introns) and repetitive sequences.

This complicates the cloning strategies, hence DNA is not preferred.

Hence m-RNA is preferred.

m-RNA is preferred for the following reasons :

m-RNA represents the actual genetic information being expressed.

Selection and isolation of m-RNA are easy.

As introns are removed during processing m-RNA reflects the coding sequence of the gene.

The synthesis of recombinant protein is much easier with m –RNA cloning.

APPLICATIONS

Essentially every area of biological research has beenaffected by the use of rDNA technology. Proteinstructure/function relationship studies and geneexpression and regulation research have been enormouslyenhanced by this powerful tool.

Transgenic animals (into which DNA from another specieshas been inserted) have been bred to expand the study ofhuman biochemical processes and diseases. Transgenicmice that are highly susceptible to breast cancer orAlzheimer's disease have furthered the understanding ofthose diseases.

Modern medicine is inextricably linked with rDNAtechnology. Gene therapy replaces defective genes withfunctional ones, delivered to the patient by way of asuitable vector, usually a disabled virus. The firstmoderately successful gene therapy was instituted to treatan inborn immune deficiency disease (ADA deficiency)caused by a defective enzyme, adenine deaminase.

Cancer research and treatments as well as some vaccinedevelopment make use of rDNA technology. Attempts tomodify animals genetically in such a way that organs suitablefor transplant into humans may be harvested are now beingmade.

Agricultural uses of recombinant DNA technology areexpanding. Genetically engineered bacteria sprayed ontostrawberries protect the strawberries from freezing.Genes that promote herbicide resistance are incorporatedinto plants so that herbicides can be used for no-tillfarming. Some plant species have been transformed byrDNA containing genes that promote resistance to insectsand pathogens.

The industrial use of rDNA technology includes theproduction of bleach-resistant enzymes that are used inlaundry detergents to degrade.

REFERENCES

Molecular biology, David freifelder, second edition, Narosa publishing house, pg.no 705-714.

Biochemistry , U . Satyanarayna , U.Chakrapani, third edition, Books and allied publications, pg.no 578-615.