GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
genetic engineering: Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. Many organism are manipulated with the help genetic engineering useful for mankind.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries will be discussed: genomic libraries and complementary DNA (cDNA) libraries.
Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome.
In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another.
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
genetic engineering: Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. Many organism are manipulated with the help genetic engineering useful for mankind.
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries will be discussed: genomic libraries and complementary DNA (cDNA) libraries.
Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome.
In contrast, cDNA libraries contain those DNA sequences that appear as mRNA molecules, and these differ from one cell type to another.
This presentation will give you an in-depth look at modern techniques and appliations of biotechnology. It will get you thinking about the potential for biotechnology to change your lives in the future. Please take Cornell Notes on the following slides.
Genetic engineering principle, tools, techniques, types and applicationTarun Kapoor
Basic principles of genetic engineering.
Study of cloning vectors, restriction endonucleases and DNA ligase.
Recombinant DNA technology. Application of genetic engineering in medicine.
Application of r DNA technology and genetic engineering in the products:
a. Interferon
b. Vaccines- hepatitis- B
c. Hormones- Insulin.
Polymerase chain reaction
Brief introduction to PCR
Basic principles of PCR
If a microbiologist is studying bacteria that premeditate, or break down, toxic wastes and wants to know which specific genes are active when that bacterium is degrading, say, PCBs, he would likely use a tool called the DNA microarray.
Microarrays enable scientists to monitor the activities of hundreds or thousands of genes at once. All microarrays (also called DNA chips or gene chips) work on the basic principle that complementary nucleotide sequences in DNA (and RNA) match up like the two halves of a piece of Velcro coming together.
Pattern of gene activity on a microarray chip.
A microarray consists of an orderly arrangement of bits of genetic material in super-tiny spots laid down in a grid on a suitable surface, often a glass slide with a specially chemically treated surface.
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.
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
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.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
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
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These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
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
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.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
5. Fifteen years ago, scientists in
Edinburgh announced to the world an
incredible breakthrough: the creation
of the first cloned animal–a sheep who
originated from a cell taken from an
adult mammal.
Dolly’s birth sparked a vigorous debate
about the controversial technique and
its potential application to humans.
5
6. DOLLY…. a female domestic sheep, and
the first mammal to be cloned from an
adult somatic cell
(5 July 1996 – 14 February 2003)
6
7. Copycat….!!!!!!!!!!!!!
The world's first cloned kitten, named Cc. It was created
by scientists in Texas using a cell taken from an adult
tortoise shell. The photo, taken on December 22 2001
when the kitten was seven weeks old, was made public
7
in February 2002.
9. Introduction
Genetic engineering is a tool of biotechnology
Sophisticated and most advanced.
Genetic Engineering includes techniques
of DNA analysis
to manipulate DNA
change DNA sequence and bring about a
desirable genetic expression.
9
10. Applications of Genetic engineering
fields of medicine, agriculture, animal
farming, ecology, paleontology, etc.
Medical applications of DNA technology
1. Basic research - understanding of
structure and functions of DNA & proteins.
2. Diagnosis of diseases - genetic and microbial.
10
11. Medical applications ………contd
3. Forensic applications
4. Production of proteins for
Replacement therapy
Disease prevention
(e.g. insulin)
(e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
5. Treatment of genetic diseases (gene therapy)
11
12. Applications in agriculture
PLANTS
1. disease-resistant and
insect-resistant, high
yielding crops
2. Hardier fruit
3. 70-75% of food in
supermarket is
genetically modified.
13. Applications in animal farming
Genetically modified
organisms are called
transgenic organisms.
1.
Mice – used to study human
immune system
2.
Chickens – more resistant to
infections
3.
Cows – increase milk supply
and leaner meat
4. Goats, sheep and pigs –
produce human proteins in
their milk.
14. Transgenic Goat
Human DNA in
a Goat Cell
.
This goat contains a human
gene that codes for a blood
clotting agent. The blood
clotting agent can be harvested
in the goat’s milk.
16. Some Important Tools of Genetic Engineering
1) Enzymes
Restriction Endonucleases (REs):
DNA ligase
DNA Polymerases
Reverse transcriptases
2)Vectors
Plasmid
Bacteriophage,
Cosmid
Yeast
17. Some Important Tools of Genetic Engineering
Enzymes
Restriction Endonucleases (REs):
used as scissors to cut DNA -DNA scissors
at specific DNA sequences
to generate a set of smaller fragments.
Genomic
DNA
DNA
fragments
17
19. DNA Polymerases
Synthesis of DNA using DNA template and
dNTPs
Reverse transcriptase
Enzyme found in retroviruses that makes
DNA copy, using RNA as template
RNA
cDNA
dsDNA
19
20. Vectors
Into the DNA of the vector a foreign DNA can
be inserted, integrated/incorporated.
Use : For amplification by cloning and for
gene therapy.
Examples : Plasmid
Bacteriophage,
Cosmid
Yeast
20
21. Plasmid
A small, circular, dsDNA
present in bacteria
Confer antibiotics resistance against the bacteria
many copies of plasmid in a bacterium
replicate independent of the bacterial DNA.
21
22. Bacteriophage
is a virus that can infect bacteria
Cosmid
plasmid + Cos sites
for binding to bacteriophages
can carry larger DNA fragments
22
23. Restriction Endonucleases (REs)
•recognize specific DNA sequences- called
“palindrome”
(restriction sites)
• Example : EcoR I (E. coli RY 13) recognises sequence
5’ GAATTC 3’.
•cuts the phosphodiester bonds of the DNA
on both the strands.
23
27. Restriction Endonucleases (REs)
•Examples:
EcoRI;
Hpa I;
BamHI;
Taq I.
REs are isolated from bacteria.
Biological function of RE in bacteria :
is to recognize and cleave foreign DNA
(e.g. DNA of an infecting virus).
27
28. Applications of REs in Genetic Engineering
1)
sequencing of DNA
2)
cloning of DNA
3)
4)
antenatal diagnosis of inherited disorders
( RFLP analysis)
DNA finger printing
(having forensic applications)
5)
for Southern blot technique
(for detecting the presence of a particular
base sequence in the sample DNA).
28
29. Some Important Techniques in DNA Analysis
and Genetic Engineering:
DNA Amplification:
production of many identical copies of a DNA
fragment of interest.
Uses
1) further DNA analysis or
2) for large-scale genetic expression
(protein production).
29
30. Types of DNA amplification
Cloning
Polymerase Chain Reaction
(PCR)
in vivo method
using bacteria
an in vitro method using
DNA polymerase
used to amplify
longer segments of
DNA
shorter segments of DNA
can be amplified
suitable for large-scale shorter time for amplifying
DNA fragments
protein production
30
31. Cloning
Production of an identical copy of
either DNA or a cell or an organism
is called cloning.
-2 Types.
1)Molecular cloning -production of
identical DNA molecules
(i.e., identical
in base-sequence)
2)Somatic cloning -production of cells or
organisms with identical genetic makeup.
31
32. DNA Cloning
Recombinant DNA Technology- Cloning a DNA
Fragment
Two principal steps :
Constructing a recombinant DNA molecule
-gene of one species is transferred to another
living organism.
-usually, a human gene is transferred to a
bacteria.
Amplifying the recombinant DNA
molecule in a bacterial host
32
36. 2. Selection, Isolation and Amplification of
Recombinant DNA:
by specific techniques
(eg. by antibiotic sensitivity technique)
and allowed to multiply in a suitable culture.
3. Release of the Cloned DNA Molecules from
the Bacteria:
by using the same RE as used for cleaving of DNA
36
37. Applications of recombinant DNA
Technology
Used in the fields of
Medicine, Agriculture, Animal
Farming, Ecology, Paleontology, etc.
37
38. Medical applications of Recombinant DNA
Technology
1.Production of proteins for
Replacement therapy
Disease prevention
(e.g. insulin)
(e.g. vaccines)
Diagnostic tests (e.g. monoclonal antibodies).
2.Treatment of genetic diseases (gene
therapy)
38
39. Production of Proteins Using Recombinant
DNA Technique :
proteins, especially human proteins
produce large amounts of proteins
provide human proteins, which are not
antigenic when administered to humans.
39
40. Proteins produced are used for:
Replacement therapy and other treatments
(e.g. insulin, growth
hormone, interleukins, antihemophilic
factor, interferon, etc.).
Disease prevention
(e.g. vaccines, such as hepatitis B antigen)
Diagnostic tests
(e.g. monoclonal antibodies).
40
41. Human insulin is
produced using
Recombinant DNA
Technique :
Recombinant insulin (Humulin)
Recombinant Human Growth Hormone
43. Polymerase Chain Reaction (PCR)
in vitro method for DNA amplification
much faster
more sensitive method than cloning.
very little DNA sample is sufficient
can only amplify short segments of DNA
cannot be used for amplifying genes and
for production of proteins
43
44. Use : To amplify a short sequence of DNA
Procedure :
(1)
A mixture of
DNA sample + dNTP’s + Primers +
Enzyme : Taq DNA polymerase
(2)
Treatment of the mixture :
1 cycle
94 - 95 C Denaturation of DNA
30 – 60 sec
52 - 54 C Annealing of primers
30 – 60 sec
72 C Extension of the DNA
1 min
44
45. Test DNA sample
Separation DNA strands
Primers anneal
1 cycle
Extension by DNAP
No. of cycles : 30 - 45
Product : Every cycle the DNA doubles
45
46. Advantages of PCR
1. Very little DNA sample is required
2. Amplification time is very short.
3. Amplification rate is high.
Applications of PCR
Useful : when insufficient DNA molecules are
present
in
test
samples
for
DNA
analytical techniques.
46
47. Uses of PCR
1. Diagnostic uses
used to quickly detect microbial
infections, when the number of microbes is
less in the sample.
Examples :Diagnosis of
Tuberculosis (TB) Mycobacterium tuberculi
AIDS
HIV
47
48. 2. Prenatal diagnosis of genetic disorders
Sections of genes, having particular
mutations known to cause a disease are
Amplified
Sequenced
Diagnosis
Example : Detection of
Sickle cell anemia (HbS)
48
49. 3. Forensic Uses:
Samples used : Blood, saliva, semen, hair
Obtained from : a victim or suspect
Volume of the sample : is insufficient
Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
i.e., DNA fingerprinting
49
50. Gene therapy : treatment of Genetic disorders .
•Severe Combined Immuno Deficiency (SCID)
Adenosine deaminase
• Cystic Fibrosis
Chloride channel
• Familial Hypercholesterolemia
Receptor for LDL
• Hemophilia
Clotting factor (factor VIII or IX)
50
51. Gene therapy
• Gene therapy is aimed at treating genetic
disorders
• involves introduction of normal foreign gene
into somatic cells of the patient having the
genetic disease to compensate for the defective
protein, which is the product of the mutant
gene.
51
52. Gene therapy-procedure
• The procedure involves,
1) isolation of the healthy gene
2) incorporation of this gene into a carrier or
vector and
3) delivering the vector into the target cells.
52
53. Gene therapy-proceedure
• isolation of the healthy gene
-done by isolating its mRNA first
-using this mRNA as a template, cDNA is
synthesised, using reverse transcriptase.
-from thic cDNA, double strande DNA is
synthesised using DNAP.
53
55. Gene therapy-proceedure..
2) incorporation
of this gene into a
carrier or vector
-Vectors used are
retroviruses, adenoviruses and
plasmid-liposome complexes.
-this is done by recombinant DNA
technology, as described earlier.
55
56. Gene therapy-proceedure….
• delivering the vector into the target
cells.
• The vector with normal gene is now introduced
to the patient.
•The cells of this patient will start producing the
normal protein which was deficient earlier.
•
Disease is cured.
56
60. Uses of DNA library
1. For Protein expression
o
Incorporation into a vector - DNA Chimera
o Cloned
Suitable bacteria
o Protein expressed
2. As a probe for analytical techniques
o Detecting specific nucleotide sequence in
test samples.
o As in Southern and Northern blot techniques.
60
61. DNA Probes are
Single stranded, fragments / pieces of DNA
Contain nucleotide sequence complimentary to
the target sequence
Radiolabeled with radioisotopes (usually 32P)
to visualize on an X-ray film
Use : for detecting a target sequence in
Southern and Northern blot techniques
61
62. Examples for probes
Synthetic oligonucleotides
RNA
Antibodies (protein) -as a probe for protein
molecule -in Western blot technique.
62
63. Blot techniques :
Analytical techniques used in
Recombinant technology
Done on test samples
Types
Detection of
Southern
DNA
Northern
RNA
Western
Protein
63
64. Applications of Blot techniques : in
• Research
• Diagnosis of diseases (microbial and genetic)
• Forensic medicine.
Southern Blot Technique
Process
:
6 steps
1) Extraction of DNA from the test sample/cells
2) Digestion by a suitable RE
– Product DNA fragments
64
65. 3) Electrophoresis of the digest
- Separation of fragments
4) Denaturation of DNA and blotting onto a
membrane (nitrocellulose membrane)
5) Adding a radiolabeled DNA probe
6) Autoradiography : Visualization on X-ray film .
DNA fragments hybridized with the
radiolabeled DNA probes.
65
67. The pattern observed on Southern blot
analysis depends on :
•
the specific RE used
•
location of the restriction site in
the DNA sample
• the probe used.
67
68. Restriction Fragment Length Polymorphism (RFLP)
Analysis
Extraction of Human chromosome
Digestion with one or more REs
Southern blot
Visualization
Application :
molecular analysis of genes involved in disease.
68
69. Medical Applications of Genetic Engineering
1. Basic research for understanding structure
and functions of DNA and proteins.
Recombinant DNA technology has made possible :
• Complete sequencing of the human genome
(Human Genome Project)
• Gene localizing and
• defining the map of the human genome.
69
70. • Isolation and detailed molecular analysis of
genes
involved
in
disease
(using
RFLP
analysis).
2. Diagnosis of diseases - genetic and microbial.
Techniques used : PCR, Southern blot & RFLP
Test sample
: Amniotic fluid
Time of Test
: Prenatal diagnosis
70
71. Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
3. Forensic Uses:
For identifying dead bodies
Settling parental disputes.
Identifying criminals.
71
72. Samples used : Blood, saliva, semen, hair
Obtained from
: a victim or suspect
Volume of the sample : is insufficient
Sample
PCR
Amplified DNA
Amplification of DNA
DNA analytical
techniques
i.e., DNA fingerprinting
72
73. 4. Production of Proteins Using Recombinant
DNA Technique :
proteins, especially human proteins
produce large amounts of proteins
provide human proteins, which are not
antigenic when administered to humans.
73
74. Proteins produced are used for:
Replacement therapy and other treatments
(e.g. insulin, growth
hormone, interleukins, antihemophilic
factor, interferon, etc.).
Disease prevention
(e.g. vaccines, such as hepatitis B antigen)
Diagnostic tests
(e.g. monoclonal antibodies).
74
75. 5. Treatment of genetic diseases :
Example :
Gene therapy
Involves
Introduction of normal foreign gene
Into somatic cells of the patient having the
genetic disease
To compensate for the defective protein,
Which is the product of the mutant gene.
75
76. Genetic disorders treated by Gene therapy
(attempt) :
•Severe Combined Immuno Deficiency (SCID)
Adenosine deaminase
• Cystic Fibrosis
Chloride channel
• Familial Hypercholesterolemia
Receptor for LDL
• Hemophilia
Clotting factor (factor VIII or IX)
76
77. 1.
2.
3.
4.
What are restriction Endonucleases? Give two examples.
(3)
What is reverse transcriptase? What is its significance?
(3)
Reverse transcriptase.
(3)
What is plasmid? What are its applications in recombinant DNA
technology?
(4)
5. Discuss in detail recombinant DNA technology and its clinical
application.
(5)
6. What is “Recombinant DNA”? Mention applications of genetic
engineering.
(1+3 =4 )
7. Describe the clinical applications of recombinant DNA technology. (4 )
8. Give two applications of recombinant DNA technology.
(3)
9. What is Polymerase Chain Reaction (PCR)? Mention application of
PCR.
(3)
10.What is polymerase chain reaction? Mention its applications
(3)
11.PCR
(4 )
12.Polymerase chain reaction
(3)
13.Gene therapy
(4)
14.What is gene therapy? Name vectors used for gene therapy. (3)
77
78. MULTIPLE CHOICE QUESTIONS
1. DNA Scissors is______________.
a) DNA Polymerase
b) 3’→5’ Exonuclease
c) Restriction endonuclease d) RNase H.
2. Two fragments of DNA are joined by ______.
a) DNA Polymerase b) DNA Ligase
c) Topoisomerase
d) Reverse transcriptase.
78
79. 3. An example for an RNA dependant DNA
polymerase is ____________.
a) DNA Polymerase b) RNA Polymerase
c) Primase
d ) Reverse transcriptase.
4. ____________ confer antibiotic resistance to
bacteria.
a) Genomic DNA
b) Mitochondria
c) Cell wall
d ) Plasmids.
79
80. 5. ____________ is an example for a recombinant
protein used in disease prevention.
a) HB antigen
b) Interleukins
c) Interferons
d ) Insulin.
. ___________ is an example for a recombinant
6.
protein used in replacement therapy.
a) HB antigen
b) Antibodies
c) Oral polio vaccine
d ) Insulin.
80
81. 7. Applications of REs in genetic engineering include
these except_____________.
a) Cloning of DNA
b) Antenatal diagnosis of inherited disorders
c) Radiolabeling
d) DNA finger printing. .
8. The function of polymerase chain reaction is to
_____________.
a) amplify DNA
b) destroy DNA
c) synthesize proteins d) confer antibiotic resistance.
81
82. 9. These can be used as a vector in DNA cloning
except
a) Plasmid
b) Cosmid
c) Oligonucleotides
d) Bacteriophage.
10. Inserting DNA fragment of interest into the DNA
of. a vector produces a molecule which is called by all
these names except
a) Recombinant DNA
b) Recombinant protein
c) DNA chimera
d) Chimeric DNA.
82
83. 11. Ideally, for cloning, both the vector and the DNA
of interest should be cleaved with the same
a) Endonuclease
b) Exonuclease
c) Restricted endonuclease d) RNase H.
12. These are advantages of PCR technique except
a) High rate of amplification
b) Less time required
c) Small amounts of test sample is needed
d) High rate of errors.
83