Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. The key steps involve choosing a host organism and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into host cells, selecting cells containing the recombinant DNA, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
Molecular Biology research evolves through the development of the technologies used for carrying them out. It is not possible to research on a large number of genes using traditional methods
Molecular Biology research evolves through the development of the technologies used for carrying them out. It is not possible to research on a large number of genes using traditional methods
Assignment on Recombinant DNA Technology and Gene TherapyDeepak Kumar
Assignment on Recombinant DNA Technology and Gene Therapy Basic principles of recombinant DNA technology-Restriction enzymes, various types of vectors, Applications of recombinant DNA technology. Gene therapy- Various types of gene transfer techniques, clinical applications and recent advances in gene therapy
GENOMIC MAPPING:FISH(Fluorescent in situ hybridization )UTTARAN MODHUKALYA
Genomic mapping is a graphic representation of thearrangement of genes or DNA sequences on chromosome & used to identify and record the location of gene & distances between genes on chromosome.
There are mainly two kinds of genome maps are known :1.Genetic or linkage maps &2. Physical maps
Where Physical map provides detail of the actual physicaldistance between genetic markers, as well as the exactlocation of genes.
An example of Physical mapping is FISH. FISH is a powerful technique for detecting RNA or DNA sequences in cells, tissues & tumors
This presentation was created by Ioanna Leontiou and it is intended as a creative and flexible tool for students on Biological sciences who focus on the chromosome segregation. It is created to facilitate students performing research projects in our lab (especially during Covid restrictions), but it is suitable for every student who wants to learn more about chromosomes and the molecular mechanism controlling chromosome segregation. The presentation includes a generic overview of the cell division, illustrates the chromosome structure and provides molecular details of the spindle assembly checkpoint, an important pathway that ensures high fedility of chromosome segregation through mitosis. It also includes an introduction to some of the molecular biology techniques used in a yeast lab and incoporates some fluorescent microscopy images/videos. At the end of the presentantion there is a list of open access scientific publications for further reading on the the molecular mechanism of spindle checkpoint and some links of some very interesting sites, which include a range of videos on laboratory molecular biology techniques, research talks and guided papers. The purpose of this presentantion is to create a piece of work that students could return to when needed. Diagramms and illustrations are also encouranged to be used by scientists, science communicators and educators.
This presentation is licensed under a Creative Common Attribution-ShareAlike 4.0 (CC BY-SA 4.0), unless otherwise stated on the specific slide.
Site directed mutgenesis, OLIGONUCLEOTIDE DIRECTED MUTAGENESIS Vipin Shukla
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In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Two approaches (clone by clone & whole genome shotgun).
Types of DNA sequencing ( 1st, next and 3rd).
Crop genomes sequenced . (Example :Arabidopsis,Rice, Pigeon pea)
Assignment on Recombinant DNA Technology and Gene TherapyDeepak Kumar
Assignment on Recombinant DNA Technology and Gene Therapy Basic principles of recombinant DNA technology-Restriction enzymes, various types of vectors, Applications of recombinant DNA technology. Gene therapy- Various types of gene transfer techniques, clinical applications and recent advances in gene therapy
GENOMIC MAPPING:FISH(Fluorescent in situ hybridization )UTTARAN MODHUKALYA
Genomic mapping is a graphic representation of thearrangement of genes or DNA sequences on chromosome & used to identify and record the location of gene & distances between genes on chromosome.
There are mainly two kinds of genome maps are known :1.Genetic or linkage maps &2. Physical maps
Where Physical map provides detail of the actual physicaldistance between genetic markers, as well as the exactlocation of genes.
An example of Physical mapping is FISH. FISH is a powerful technique for detecting RNA or DNA sequences in cells, tissues & tumors
This presentation was created by Ioanna Leontiou and it is intended as a creative and flexible tool for students on Biological sciences who focus on the chromosome segregation. It is created to facilitate students performing research projects in our lab (especially during Covid restrictions), but it is suitable for every student who wants to learn more about chromosomes and the molecular mechanism controlling chromosome segregation. The presentation includes a generic overview of the cell division, illustrates the chromosome structure and provides molecular details of the spindle assembly checkpoint, an important pathway that ensures high fedility of chromosome segregation through mitosis. It also includes an introduction to some of the molecular biology techniques used in a yeast lab and incoporates some fluorescent microscopy images/videos. At the end of the presentantion there is a list of open access scientific publications for further reading on the the molecular mechanism of spindle checkpoint and some links of some very interesting sites, which include a range of videos on laboratory molecular biology techniques, research talks and guided papers. The purpose of this presentantion is to create a piece of work that students could return to when needed. Diagramms and illustrations are also encouranged to be used by scientists, science communicators and educators.
This presentation is licensed under a Creative Common Attribution-ShareAlike 4.0 (CC BY-SA 4.0), unless otherwise stated on the specific slide.
Site directed mutgenesis, OLIGONUCLEOTIDE DIRECTED MUTAGENESIS Vipin Shukla
INTRODUCTION, HISTORY, MUTATION, DIRECTED MUTAGENESIS,BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS,METHOD FOR SITE DIRECTED MUTATIONS,THE SINGLE PRIMER METHOD, CASETTEE MUTAGENESIS, PCR-SITED DIRECTED MUTAGENESIS, APPLICATION OF SITE DIRECTED MUTAGENESIS.
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Two approaches (clone by clone & whole genome shotgun).
Types of DNA sequencing ( 1st, next and 3rd).
Crop genomes sequenced . (Example :Arabidopsis,Rice, Pigeon pea)
Genetic Engineering, also called as recombinant DNA technology, involves the group of techniques used to cut up and join together genetic material, especially DNA from different biological species, and to introduce the resulting hybrid DNA into an organism in order to form new combinations of heritable genetic material. This slide will illustrate the basic concepts and steps involved in Genetic Engineering.
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Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
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Dna cloning
1. BY :
SYED FASIH UDDIN
M.PHARMACY
12111S0108
PHARMACOLOGY
SHADAN COLLEGE OF PHARMACY
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.