BY :

SYED FASIH UDDIN

M.PHARMACY

12111S0108

PHARMACOLOGY

SHADAN COLLEGE OF PHARMACY

Submitted To

IntroductionThe terms "recombinant DNA

technology," "DNA cloning," "molecular cloning"or "gene cloning" all refer to the same process.

CLONONG refers to the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing identical DNA molecules.

DNA CLONING is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.

Molecular cloning methods are central to many contemporary areas of modern biology and medicine. The structure of part of

DNA double helix

HistoryPrior to the 1970s, the understanding of genetics and

molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms.

This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleases, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered.

The first recombinant DNA molecules were generated and studied in 1972..

STEPS INVOLVED IN MOLECULAR CLONINGChoice of host organism and cloning

vector.Preparation of vector DNA.Preparation of DNA to be cloned.Creation of recombinant DNA.Introduction of recombinant DNA into host

organism.Selection of organisms containing

recombinant DNA.Screening for clones with desired DNA

inserts and biological properties.

Choice of host organism and cloning vector: Although a very large number of host organisms and molecular

cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.

Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility..

(1) an origin of DNA replication is necessary for the vector (and recombinant sequences linked to it) to replicate inside the host organism.

(2) one or more unique restriction endonuclease recognition sites where foreign DNA may be introduced.

(3) a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences.

(4) an additional gene that can be used for screening which cells contain foreign DNA.

Preparation of vector DNA:

The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.

The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA.

Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI.

Preparation of DNA to be cloned:For cloning of genomic DNA,

the DNA to be cloned is extracted from the organism of interest.

The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography)

The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector

Creation of recombinant DNA with DNA ligase:The creation of recombinant DNA is in many ways the

simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.

Introduction of recombinant DNA into host organism:

The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g.transformation, transduction, transfection, electroporation).

When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation.

 In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.

In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection.

Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present)

Selection of organisms containing vector sequences:Cells that have not taken up DNA

are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.

When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin.

Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. 

Screening for clones with desired DNA inserts and biological properties:

Modern bacterial cloning vectors (e.g. pUC19 and later derivatives including the pGEM vectors) use the blue-white screening system to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted).

In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work.

 Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing recombinant plasmids remain colorless (white).

Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.

The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library

TYPES OF CLONING:FUNCTIONAL EXPRESSION OF CLONING:

• It focuses on obtaining a specific cDNA of known function. There are many variations on this approach but they all rely on the ability to search for and isolate cDNAs based functional activitye.g.,the electrophysiological measurement of ion conductance's following expression of cDNAs in frog oocyte.

• The advantage of functional expression cloning is that it does not rely on knowledge of the primary amino acid sequence. This is a definite advantage when to clone proteins of low abundance.

POSITIONAL CLONING: It can be used to localize fragments of DNA representing

genes prior to isolating the DNA.

An e.g. of the use of positional cloning is the cloning of gene responsible for Cysctic fibrosis (CF).By studying the patterns of inheritance of the disease and then comparing this with known chromosomal marker (linkage analysis) it was possible to locate the gene on human chromosome 7.

The advantage is that it also does not require the specific knowledge of protein and can provide imp new biological targets for drug development and the treatment of diseases.

HOMOLOGY-BASED CLONING:• It involves use of previously cloned genes to guide

identification and cloning of evolutionary related genes.

• It tales the advantage of the fact that nucleotide sequences encoding imp functional domains of proteins tend to be conserved during the process of evolution. Thus nucleotide sequences encoding regions involved with enzymatic activity can be used as a probe that will hybridize to complementary nucleotide sequences that may be present on other genes that have similar enzymatic activity .

Advantages:• It can be used to identify families of related genes, does not

rely on functional activity of given protein.• Can provide novel targets for drug discovery.

APPLICATIONS OF CLONING:

Genome organization and gene expression:

Molecular clones are used to generate probes that are used for examining how genes are expressed, and how that expression is related to other processes in biology. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate the genes, or make more subtle mutations using regional mutagenesis or site-directed mutagenesis.

Production of recombinant proteins:

Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein.

Many useful proteins are currently available as recombinant products like: (1)Medically useful proteins whose administration can correct a defective or

poorly expressed gene (e.g. recombinant factor VIII, a blood-clotting factor deficient in some forms of hemophilia, and recombinant insulin, used to treat some forms of diabetes.

(2)Proteins that can be administered to assist in a life threatening emergency (e.g. tissue plasminogen activator, used to treat strokes.

(3) Recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g. hepatitis B vaccine)

(4) Recombinant proteins as standard material for diagnostic laboratory tests.

Transgenic organisms:Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed genetically modified organisms (GMOs).Although most GMOs are generated for purposes of basic biological research ( for example, transgenic mouse), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (pharming), herbicide-resistant crop plants, and fluorescent tropical fish (GloFish) for home entertainment.

Gene therapy:

• Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease.

• Gene therapy can be broadly divided into two categories.• The first is alteration of germ cells, that is, sperm or eggs, which

results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings.

• The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient.

• Treatment of cancers and blood, liver, and lung disorders was achieved.

QUERIES