Gene cloning involves producing exact copies of a particular gene or DNA sequence using genetic engineering techniques. The key steps are: 1) isolating the gene of interest from an organism's DNA, 2) inserting the gene fragment into a vector to create recombinant DNA, and 3) introducing the recombinant DNA into a host cell where it can be replicated to produce multiple copies of the gene. Common vectors used are plasmids and bacteriophages, while bacterial cells are frequently used as hosts. The multiplied gene copies can then be isolated and purified. Gene cloning has applications such as determining gene sequences, identifying gene functions, and engineering organisms for useful purposes like insulin production.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
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.
Transfection methods (DNA to host cell) Erin Davis
Transfection of DNA to host cell can be done by various methods in lab scale.Gene gun,electroporation,lipofection .These methods are used to transfer DNA to the host cell.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
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.
Transfection methods (DNA to host cell) Erin Davis
Transfection of DNA to host cell can be done by various methods in lab scale.Gene gun,electroporation,lipofection .These methods are used to transfer DNA to the host cell.
Prokaryotic and eukaryotic gene structurestusharamodugu
Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA).
Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA). Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA).
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
Isolation and Purification of Chromosomal DNA,Plasmid DNA,Bacteriophage DNA used in Recombinant DNA Technology or Biotechnology to produce Recombinant DNA or Desired DNA
Prokaryotic and eukaryotic gene structurestusharamodugu
Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA).
Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA). Organization of genome in Prokaryotes:
The term prokaryote means “primitive nucleus”. Cell in prokaryotes have no nucleus. The prokaryotic chromosome is dispersed within the cell and is not enclosed by a separate membrane. Much of the information about the structure of DNA comes from studies of prokaryotes, because they are less complex than eukaryotes. Prokaryotes are monoploids they have only one set of genes (one copy of the genome). In most viruses and prokaryotes, the single set of genes is stored in a single chromosome (single molecule either RNA or DNA).
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
INTRODUCTION
ROLE IN CELL LINE CHARACTERIZATION
CAUSES OF TRANSFORMATION
METHODS OF TRANSFECTION
CHARACTERISTICS OF TRAANSFORMED CELLS
GENETIC INSTABILITY
IMMORTALIZATION
ABRERANT GROWTH CONTROL
TUMORIGENECITY
CHROMOSOMAL ABERATION
APPLICATION
CONCLUSION
REFERENCE
Isolation and Purification of Chromosomal DNA,Plasmid DNA,Bacteriophage DNA used in Recombinant DNA Technology or Biotechnology to produce Recombinant DNA or Desired DNA
Gene Cloning has brought a revolutionary change in the fields of Genetic Engineering and Biotechnology. Application of this technique has become an increasingly important tool in molecular research due to its simplicity, cost effectiveness, rapidity, and reliability.
DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid.- [https://www.khanacademy.org/science/...dna.../dna-cloning.../a/overview-dna-cloning]
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.
Cloning is a technique scientists use to make exact genetic copies of living things. Genes, cells, tissues, and even whole animals can all be cloned. Some clones already exist in nature. Single-celled organisms like bacteria make exact copies of themselves each time they reproduce.
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
gene cloning, secreening a library, cloning products, requrements, aqsa ijaz
Recombinant DNA molecules are only useful if they can be made to replicate and produce a large number of copies. A typical gene-cloning procedure includes the following steps (See Campbell, Figure 19.3):
Step 1: Isolation of two kinds of DNA.
Bacterial plasmids and foreign DNA containing the gene of interest are isolated.
In this example, the foreign DNA is human, and the plasmid is from E. coli and has two genes:
--> ampR that confers antibiotic resistance to ampicillin.
--> lacZ that codes for beta-galactosidase, the enzyme that catalyzes the hydrolysis of lactose
Note that the recognition sequence for the restriction enzyme used in this example is within the lacZ gene.
Step 2: Treatment of plasmid and foreign DNA with the same restriction enzyme.
The restriction enzyme cuts plasmid DNA at the restriction site, disrupting the lacZ gene.
The foreign DNA is cut into thousands of fragments by the same restriction enzyme; one of the fragments contains the gene of interest.
When the restriction enzyme cuts, it produces sticky ends on both the foreign DNA fragments and the plasmid.
Step 3: Mixture of foreign DNA with chopped plasmids.
Sticky ends of the plasmid will base pair with complementary sticky ends of foreign DNA fragments.
Step 4: Addition of DNA ligase.
DNA ligase catalyzes the formation of covalent bonds, joining the two DNA molecules and forming a new plasmid with recombinant DNA.
Step 5: Introduction of recombinant plasmid into bacterial cells.
the naked DNA is added to a bacterial culture.
Some bacteria will take up the plasmid DNA by transformation.
Step 6: Production of multiple gene copies by gene cloning and selection process for transformed cells.
Bacteria with the recombinant plasmid are allowed to reproduce, cloning the inserted gene in the process.
Recombinant plasmids can be identified by the fact that they are ampicillin resistant and will grow in the presence of ampicillin.
Step 7: Final screening for transformed cells.
X-gal, a modified sugar added to the culture medium, turns blue when hydrolyzed by beta-galactosidase. It is used as an indicator that cells have been transformed by plasmids containing the foreign insert.
Since the foreign DNA insert disrupts the lacZ gene, bacterial colonies that have successfully acquired the foreign DNA fragment will be white. Those bacterial colonies lacking the DNA insert will have a complete lacZ gene that produces beta-galactosidase and will turn blue in the presence of X-gal.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2. • The production of exact copies of a
particular gene or DNA sequence using genetic engineering
techniques is called gene cloning.
• The term “gene cloning,” “DNA cloning,” “molecular cloning,”
and “recombinant DNA technology” all refer to same technique.
• When DNA is extracted from an organism, all its genes are
obtained. In gene (DNA) cloning a particular gene is copied
forming “clones”.
• Cloning is one method used for isolation and amplification of
gene of interest.
• DNA cloning can be achieved by two different methods:
• Cell based DNA cloning
• Cell-free DNA cloning bypolymerase chain reaction (PCR)
3. • Principle of Gene Cloning
• A fragment of DNA, containing the gene to be cloned, is inserted into a
suitable vector, to produce a recombinant DNA molecule. The vector acts as a
vehicle that transports the gene into a host cell usually a bacterium, although
other types of living cell can be used. Within the host cell the vector
multiplies, producing numerous identical copies not only of itself but also of
the gene that it carries. When the host cell divides, copies of the recombinant
DNA molecule are passed to the progeny and further vector replication takes
place. After a large number of cell divisions, a colony, or clone, of identical
host cells is produced. Each cell in the clone contains one or more copies of
the recombinant DNA molecule; the gene carried by the recombinant
molecule is now said to be cloned.
4.
5. • Requirements for Gene Cloning (Cell-based)
• DNA fragment containing the desired genes to be
cloned.
• Restriction enzymes and ligase enzymes.
• Vectors – to carry, maintain and replicate cloned
gene in host cell.
• Host cell– in which recombinant DNA can
replicate.
6. • Steps in Gene Cloning
• The basic 7 steps involved in gene cloning are:
• Isolation of DNA [gene of interest] fragments to be cloned.
• Insertion of isolated DNA into a suitable vector to form
recombinant DNA.
• Introduction of recombinant DNA into a suitable organism
known as host.
• Selection of transformed host cells and identification of the
clone containing the gene of interest.
• Multiplication/Expression of the introduced Gene in the host.
• Isolation of multiple gene copies/Protein expressed by the gene.
• Purification of the isolated gene copy/protein
7. A. Isolation of the DNA fragment or gene
The target DNA or gene to be cloned must be first isolated. A gene of
interest is a fragment of gene whose product (a protein, enzyme or a
hormone) interests us. For example, gene encoding for the hormone
insulin.
The desired gene may be isolated by using restriction endonuclease
(RE) enzyme, which cut DNA at specific recognition nucleotide
sequences known as restriction sites towards the inner region (hence
endonuclease) producing blunt or sticky ends.
Sometimes, reverse transcriptase enzyme may also be used which
synthesizes complementary DNA strand of the desired gene using its
mRNA.
8. • Selection of suitable cloning vector
• The vector is a carrier molecule which can carry the gene
of interest (GI) into a host, replicate there along with the
GI making its multiple copies.
• The cloning vectors are limited to the size of insert that
they can carry. Depending on the size and the application
of the insert the suitable vector is selected.
• The different types of vectors available for cloning are
plasmids, bacteriophages, bacterial artificial
chromosomes (BACs), yeast artificial chromosomes
(YACs) and mammalian artificial chromosomes
(MACs).
• However, the most commonly used cloning vectors include
plasmids and bacteriophages (phage λ) beside all the other
available vectors.
9. • Essential Characteristics of Cloning Vectors
• All cloning vectors are carrier DNA molecules. These
carrier molecules should have few common features in
general such as:
• It must be self-replicating inside host cell.
• It must possess a unique restriction site for RE enzymes.
• Introduction of donor DNA fragment must not interfere
with replication property of the vector.
• It must possess some marker gene such that it can be used
for later identification of recombinant cell (usually an
antibiotic resistance gene that is absent in the host cell).
• They should be easily isolated from host cell.
10. • D. Formation of Recombinant DNA
• The plasmid vector is cut open by the same RE enzyme used for isolation
of donor DNA fragment.
• The mixture of donor DNA fragment and plasmid vector are mixed
together.
• In the presence of DNA ligase, base pairing of donor DNA fragment and
plasmid vector occurs.
• The resulting DNA molecule is a hybrid of two DNA molecules – the GI
and the vector. In the terminology of genetics this intermixing of different
DNA strands is called recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant DNA
molecule and the technology is referred to as the recombinant DNA
technology
11. • E. Transformation of recombinant vector into suitable host
• The recombinant vector is transformed into suitable host cell
mostly, a bacterial cell.
• This is done either for one or both of the following reasons:
– To replicate the recombinant DNA molecule in order to get
the multiple copies of the GI.
– To allow the expression of the GI such that it produces its
needed protein product.
• Some bacteria are naturally transformable; they take up the
recombinant vector automatically.
• For example: Bacillus, Haemophillus, Helicobacter pylori,
which are naturally competent.
• Some other bacteria, on the other hand require the incorporation
by artificial methods such as Ca++ ion treatment, electroporation,
etc.
12. • F.Isolation of Recombinant Cells
• The transformation process generates a mixed population of
transformed and non-trans- formed host cells.
• The selection process involves filtering the transformed host
cells only.
• For isolation of recombinant cell from non-recombinant cell,
marker gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different marker
gene (Ampicillin resistant gene and Tetracycline resistant gene.
When pst1 RE is used it knock out Ampicillin resistant gene
from the plasmid, so that the recombinant cell become sensitive
to Ampicillin.
13. • G. Multiplication of Selected Host Cells
• Once transformed host cells are separated by the screening
process; becomes necessary to provide them optimum
parameters to grow and multiply.
• In this step the transformed host cells are introduced into fresh
culture media .
• At this stage the host cells divide and re-divide along with the
replication of the recombinant DNA carried by them.
• If the aim is obtaining numerous copies of GI, then simply
replication of the host cell is allowed. But for obtaining the
product of interest, favourable conditions must be provided such
that the GI in the vector expresses the product of interest.
14. • H. Isolation and Purification of the Product
• The next step involves isolation of the multiplied GI attached
with the vector or of the protein encoded by it.
• This is followed by purification of the isolated gene
copy/protein.
15. • Applications of Gene Cloning
• A particular gene can be isolated and its nucleotide
sequence determined
• Control sequences of DNA can be identified & analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects related to
specific diseases Organisms can be ‘engineered’ for
specific purposes, e.g. insulin production, insect resistance,
etc