Recombinant DNA technology allows for the isolation, cloning, and manipulation of genes. Two key advances enabled this field: genetic engineering using restriction enzymes to isolate and modify genes in vitro, and DNA sequencing to determine the order of nucleotides. Recombinant DNA is generated by joining DNA from different sources, and molecular cloning produces large quantities of a particular DNA fragment through construction of a recombinant vector, introduction into a host cell, selective propagation of cells containing the vector, and extraction of the cloned DNA.
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
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.
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.
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.
In humans, approximately 25,000 genes exit among the 3 billion base pairs of DNA in the genome.
To study anyone of these genes, a researcher first isolates it from all of the other genes in an organisms DNA.
One isolation method has a relatively long history and involves the construction of a DNA library
When a gene is identified and copied, it is said to have been “cloned”
توثيق مراجع البحث العلمي على أنو: "إثبات ادلصادر البيانات وادلعلومات ونسبها إىلnedalalazzwy
عترب البحث العلمي ادلمنهج ذاك الذي يستويف يف مجيع مراحلو مراعاة معايري البحث العلمي ادلنهجي
خاصة فيما خيص األمانة يف اعتماد ادلراجع سواء كانت دراسات سابقة او مراجع لبعض االقتباسلت والعبارات
وزبتلف عملية التوثيق للمراجع باختالف مصدرىا ونوعها واختالف رلال زبصصها فتوثيق التت ملال خيتلف
عنو يف توثيق ادلقاالت الصحفية وخيتلف عن توثيق ادلواد االلتًتونية وىذه األخرية خيتلف يف توثيقها تبعا ألنواعها
ىي األخرى واذلدف من ذلك ىو حفاظ الباحث على سهولة العودة اىل ادلصادر وادلراجع ادلستخدمة بالنسبة
لقراء حبقو العلمي وىو أيضا من باب األمانة العلمية
A single nucleotide polymorphism (abbreviated SNP, pronounced snip) is a genomic variant at a single base position in the DNA. Scientists study if and how SNPs in a genome influence health, disease, drug response and other traits.
In humans, approximately 25,000 genes exit among the 3 billion base pairs of DNA in the genome.
To study anyone of these genes, a researcher first isolates it from all of the other genes in an organisms DNA.
One isolation method has a relatively long history and involves the construction of a DNA library
When a gene is identified and copied, it is said to have been “cloned”
توثيق مراجع البحث العلمي على أنو: "إثبات ادلصادر البيانات وادلعلومات ونسبها إىلnedalalazzwy
عترب البحث العلمي ادلمنهج ذاك الذي يستويف يف مجيع مراحلو مراعاة معايري البحث العلمي ادلنهجي
خاصة فيما خيص األمانة يف اعتماد ادلراجع سواء كانت دراسات سابقة او مراجع لبعض االقتباسلت والعبارات
وزبتلف عملية التوثيق للمراجع باختالف مصدرىا ونوعها واختالف رلال زبصصها فتوثيق التت ملال خيتلف
عنو يف توثيق ادلقاالت الصحفية وخيتلف عن توثيق ادلواد االلتًتونية وىذه األخرية خيتلف يف توثيقها تبعا ألنواعها
ىي األخرى واذلدف من ذلك ىو حفاظ الباحث على سهولة العودة اىل ادلصادر وادلراجع ادلستخدمة بالنسبة
لقراء حبقو العلمي وىو أيضا من باب األمانة العلمية
A single nucleotide polymorphism (abbreviated SNP, pronounced snip) is a genomic variant at a single base position in the DNA. Scientists study if and how SNPs in a genome influence health, disease, drug response and other traits.
Mycology is the branch of biology concerned with the study of fungi, including their genetic and biochemical properties, their taxonomy and their use to humans, including as a source for tinder, traditional medicine, food, and entheogens, as well as their dangers, such as toxicity or infection.
Rabies virus, scientific name Rabies lyssavirus, is a neurotropic virus that causes rabies in humans and animals. Rabies transmission can occur through the saliva of animals and less commonly through contact with human saliva. Rabies lyssavirus, like many rhabdoviruses, has an extremely wide host range.
Immunofluorescence (IF) is a technique that permits visualization of virtually many components in any given tissue or cell type. This broad capability is achieved through combinations of specific antibodies tagged with fluorophores. Consequently, the pos
fastidious organism is any organism that has complex or particular nutritional requirements. In other words, a fastidious organism will only grow when specific nutrients are included in its medium.
An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune system does not recognize the substance, and is trying to fight it off. An antigen may be a substance from th
Multiplex PCR is a technique whereby PCR is used to amplify several different DNA sequences simultaneously. It is a type of target enrichment approach. It was first described in 1988 as a method to detect deletion mutations in the dystrophin gene – the largest known human gene
Radio Immuno Assay, Immuno Fluorescent Test, Lab 4.pptxnedalalazzwy
A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone .
What is enzyme-linked immunosorbent assay?
A laboratory technique that uses antibodies linked to enzymes to detect and measure the amount of a substance in a solution, such as serum. The test is done using a solid surface to which the antibodies and other molecules stick.
Infectious diseases can be viral, bacterial, parasitic or fungal infections. There's also a rare group of infectious diseases known as transmissible spongiform encephalopathies (TSEs).
Classification of medical parasitology Lec.2.pptxnedalalazzwy
Parasitology is the scientific discipline concerned with the study of the biology of parasites and parasitic diseases, including the distribution, biochemistry, physiology, molecular biology, ecology, evolution and clinical aspects of parasites, including the host response to these agents.
What is toxoplasmosis? Toxoplasmosis is an infection caused by a single-celled parasite called Toxoplasma gondii. While the parasite is found throughout the world, more than 40 million people in the United States may be infected with the Toxoplasma parasite.
Integrons are genetic elements that contain a site-specific recombination system able to integrate, express and exchange specific DNA elements, called gene cassettes. 5. The complete integron is not considered to be a mobile element as such as it lacks functions for self-mobility.
Mycoplasma pneumoniae are bacteria that can cause illness by damaging the lining of the respiratory system (throat, lungs, windpipe). People can have the bacteria in their nose or throat at one time or another without being ill. People spread Mycoplasma pneumoniae bacteria to others by coughing or sneezing.
A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene.
A cell cycle is a series of events that takes place in a cell as it grows and divides. A cell spends most of its time in what is called interphase, and during this time it grows, replicates its chromosomes, and prepares for cell division. The cell then leaves interphase, undergoes mitosis, and completes its division.
Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail.
Assay of sodium hydroxide solution.pptxnedalalazzwy
sodium hydroxide is useful for its ability to alter fats. It is used to make soap and as a main ingredient in household products such as liquid drain cleaners. Sodium hydroxide is usually sold in pure form as white pellets or as a solution in water.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
2. Recombinant DNA technology
Two primary advances helped in the new era of molecular
genetics in the late 1970s to the early 1980s:
(1) The use of recombinant DNA technology (genetic
engineering) used to isolate and manipulate genes in
vitro in order to endow cells with new synthetic
capabilities (restriction enzymes)
(2) The ability to synthesize and determine the linear
order of nucleotides of DNA molecules (DNA
sequencing).
3. Advances of DNA technology
Recombinant DNA technology has made it possible
to:
• Clone (isolate and make copies of individual
genes).
• Transfer genes between bacterial species and strains
or from eukaryotes into bacteria (or vice versa).
• Causing the engineered cells to produce, sometimes
in relatively large quantities, proteins of great
economic importance such as enzymes (e. g. ,
amylases, proteases), hormones (e. g. , insulin,
growth hormone).
4. Recombinant DNA
• Recombinant DNA is
generated by covalently
joining DNA molecules
from different sources.
• The technology associated
with the construction
application of recombinant
DNA is referred to as
genetic engineering.
5.
6. Molecular coloning
• Molecular cloning is an in vivo technique for the
production of large quantities of a particular DNA
fragment.
• It contains four major steps:
1. Construction of recombinant vector.
2. Induction of the recombinant vector into suitable
host cell.
3. Selective propagation of cells containing the
vector(cloning).
4. Extraction and purification of the cloned DNA
7. Construction of recombinant
vector, which involve cutting,
modifying and joining donor
and vector DNA in vitro.
Induction of the recombinant
vector into suitable host cell.
Extraction and purification of
the cloned DNA
Selective propagation of cells
containing the vector(cloning).
8. Restriction Endonucleases
Using restriction enzymes II to create recombinant
DNA
• Cutting DNA by using restriction enzymes is
one of the most common Molecular Biology
techniques. The cut ends can be joined using
DNA ligase.
• The availability of pure restriction enzymes was
one of the first major advances in the new
science of Molecular Biology.
9. Restriction Endonucleases
restriction enzymes
• These enzymes occur naturally in bacteria.
• Enzymes that “cut” DNA in a sequence-specific
manner. recognition sequence
• Serve as a natural defense mechanism for bacteria
against viral infection (bacteriophage).
• Bacteria protect their DNA from cutting by their own
enzymes through methylation.
10. • The simplest way of cloning is to cut the donor
and the vector DNA with the same enzyme and
then join them with ligase.
11. Examples
Enzyme Recognition sequence
• EcoRI GAATTC
• HindIII AAGCTT
• BamHI GGATCC
• EcoRV GATATC
• Recognition sequences are usually 4-8 base
pairs in length and are usually palindromic
13. Cloning vectors
• DNA fragment does not contain origin of
replication (or a replicon).
• It should be joined to a replicon (vector).
• Those include : Plasmids, Viruses and
chromosomes.
14. An ideal cloning vector should be:
1. Episomal (do not integrate to the host genome,
can be separated easily).
2. Replicate autonomously giving high copy
number.
3. Allow the identification of DNA-carrying
vector those lacking them.
4. Allow the identification of cells (host) crying
DNA-carrying vector.
5. Should maintain characters that enable them to
be used in applications after cloning.
15. • Plasmids and bacteriophages are considered as
naturally poor vectors. So they are
manipulated by entering restriction sites
without affecting the sequence of the plasmid
or bacteriophages.
1. Plasmids
• Naturally occurring in bacteria.
• Used to copy already present copy of a gene
in the genomic library (sub-cloning).
• Can be used in post transcriptional activities
(small and easy to deal with).
16. 2- Bacteriophage λ:
• Used in DNA libraries.
• Can contain larger inserts
• Can be stored for long periods.
3- Cosmids
• Vectors constructed from both plasmids and bacteriophages
• Can contain large inserts thats why used in genomic libraries.
4-Phagemids:
• Plasmids that contain the OriC of the M13 Bacteriophage.
Thus can produce single stranded DNA ( can be used for
sequencing, probe synthesis and mutagenesis)
5-phasmids:
Used in post-transcriptional applications.
6-Artificail chromosomes
Can carry large DNA sequences
YACs
BACs
17. Types of Cloning Vector
Types size of cloned DNA (kb)
Plasmid 20
Lambda Phage 25
Cosmid 45
P1 phage 100
BAC 300
YAC 1000
18.
19. ex. pBR322
• Is a plasmid and was the first
widely-used E. coli cloning vectors.
• Created in 1977 in the laboratory
of Herbert Boyer (University of
California San Francisco).
• It was named after the Mexican
postdoctoral researchers who
constructed it (p stands for
"plasmid," and BR for "Bolivar"
and "Rodriguez.“
• pBR322 is 4361 base pairs in
length and contains the replicon of
plasmid pMB1.
20.
21.
22. DNA ligases
• DNA ligases catalyze formation of a phosphodiester bond
between nucleotides.
• This enzyme is used to covalently link or ligate fragments of
DNA together.
• Most commonly, the reaction involves ligating a fragment of
DNA into a plasmid vector, which is a fundamental technique
in recombinant DNA work.
• One of the most used enzymes to ligate DNA fragments is T4
DNA ligase, which originates from the T4 bacteriophage
23. DNA transfer to the cloning host
• Once recombinant vector has been constructed in
vitro it should be introduce into a host cell.
• E.coli is the major host cells in general, however
it can not introduce the DNA naturally into the
cell. To over come that problem:
• 1- Electroporation (electron transformation
through high voltatge).
• 2-Heat shock
• 3-Treatment with calcium ions Ca++.
24.
25. Vector and recombinant selection
Construction of recombinant DNA and transfection
are not always 100% successful.
Vector selection (test transfection):
• Markers: Antibiotic resistance marker.
Only the colonies that containing the plasmid can
grow in a medium containing that antibiotic.
Selection of recombinant DNA(test recombination):
• Blue- white selection:
Insert disrupts a marker (gene) that turns the cell into
blue if not distrusted hence has white color if
disrupted.
28. Recovery of cloned DNA
• The cells are cultured in solid media.
• The colonies that contain the insert are selected by
the previous methods.
• Transferred into a liquid media to generate large
quantities from it.
• Collection of host cells to re-extract the plasmids:
• Using cloning we can generate libraries for genes or
parts of genes that contain thousands of copies
29. DNA Library
1. Genomic libraries:
Collection of DNA sequences from a living
organism that has been copied into a vector
so as to be used and stored and analyzed.
2. Complementary DNA (cDNA) libraries.
Only coding parts of the genome
30. How to construct a genomic library
• The construction of a genomic library begins with cleaving
the genome into small pieces by a restriction endonuclease.
• These genomic fragments are then either cloned into
vectors & introduced into a microbe or packed into phage
particles that are used to infect the host.
• At the end many thousands of different clones each with a
different genomic DNA insert –are created.
• Therefore each clone will act as a “book” in this “library”
of DNA fragments.
• If the genomic library has been inserted into a microbe that
expresses the foreign gene, it may be possible to assay each
clone for a specific protein or phenotype
31. Digestion of the chromosomal DNA
with restriction endonuclease
Production of cDNA
(complimentary DNA)
Genomic Library
Insertion of each DNA fragment into
vector (recombinant DNA)
Transformation of Bacteria
using the recombinant vectors
Cloning of
the bacterial cells
Each clone produced is a “book”
in the “Library” of DNA fragments
cDNA Library
Extraction of mRNA
Extraction of chromosomal
DNA
Insertion of each cDNA into vector
(recombinant DNA)
Transformation of bacteria
Cloning of each recombinant
Production of Bacterial cell (clones)
containing the gene of interest