Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. It involves isolating a gene or DNA fragment and inserting it into a host organism to generate multiple copies. The key steps include choosing a host and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into a host, selecting clones containing the insert, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
1. BY :
SYED FASIH UDDIN
M.PHARMACY
12111S0108
PHARMACOLOGY
SHADAN COLLEGE OF PHARMACY
Submitted
To
2. Introduction
The 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
3. History
Prior 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..
4.
5. STEPS INVOLVED IN MOLECULAR CLONING
Choice 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.
6. 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.
7. 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.
8. 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
9. 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.
10. 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)
11. 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.
12. 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
13. 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 activity
e.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.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
