This document provides an outline for a lecture on health biotechnology. It begins with an introduction to the topic and lists various course contents that will be covered, including molecular basis of disease, genetic markers, detection of infectious agents, immunization techniques, organ transplantation, applications of transgenic animals, drug delivery systems, and uses of stem cell technology. Reference books on medical biotechnology and health and pharmaceutical biotechnology are also provided. The document then provides an outline of the lecture, discussing introduction to health biotechnology, its applications in areas like drug production, pharmacogenomics, and gene therapy. It also discusses genetic testing.
this helps to understand the normal techniques related to biotechnology in a simple manner and provides you broad idea about the subject. A brief knowledge about the topic is presented in this presentation.
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.
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
this helps to understand the normal techniques related to biotechnology in a simple manner and provides you broad idea about the subject. A brief knowledge about the topic is presented in this presentation.
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.
Introduction
Definition
History
Why are the transgenic animals being produced
Transgenic mice
Mice: as model organism
Methods of creation of transgenic mice
knock-out mice
Application of transgenic mice
Conclusion
References
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
History of Genetic Engineering
Tools of Genetic Engineering
Principles of rDNA technology
Applications of Genetic Engineering in agriculture medicine and orthodontics
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
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.
History of Genetic Engineering
Tools of Genetic Engineering
Principles of rDNA technology
Applications of Genetic Engineering in agriculture medicine and orthodontics
in this presentation, what are the steps and strategies involved the gene cloning and i was focused only on the 1st two steps of gene cloning.they are generation of foreign DNA molecules and selection of suitable vectors.
Vector mediated gene transfer methods for transgenesis in Plants.Akshay More
Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.
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.
This power point presentation has described role of biotechnology in management of diseases. This presentation also contains different biotechnological techniques for Treating, Diagnosing, preventing and understanding the disease.
INTRODUCTION
DNA VACCINES
GENE THERAPY
TIME LINE OF DEVELOPING GENE THERAPY
GENE THERAPY STRATEGIES
TECHNOLOGY OF CLASSICAL GENE THERAPY
PRINCIPLES OF GENE TRANSFER
VECTORS
VIRAL VECTORS
NON-VIRAL VECTORS
APPLICATIONS OF GENE THERAPY
ETHICAL IMPLICATIONS
THE FUTURE
CONCLUSION
REFERENCES
INTRODUCTION OF GENE THERAPY, HISTORY OF GENE THERAPY, Process of gene therapy, Methods of gene therapy, Ex vivo gene therapy , In Vivo Gene Therapy , Uses of gene therapy, Target sites for Gene Therapy , Vectors for gene therapy , Viral Vectors, Non Viral Vectors,
This presentation focuses on the science of Gene Therapy, the techniques of germ-line and somatic gene therapy and the mechanism of curing diseases and disorders using gene therapy. The presentation starts by discussing some common basic terms from genetics and moves on to the historical development of gene therapy techniques in chronological order. The different types of gene therapy techniques and their mechanisms have been discussed in detail subsequently. In concluding slides, some commercially available gene therapy products are mentioned and challenges of gene-therapy techniques have been highlighted.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
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Anti ulcer drugs and their Advance pharmacology ||
Anti-ulcer drugs are medications used to prevent and treat ulcers in the stomach and upper part of the small intestine (duodenal ulcers). These ulcers are often caused by an imbalance between stomach acid and the mucosal lining, which protects the stomach lining.
||Scope: Overview of various classes of anti-ulcer drugs, their mechanisms of action, indications, side effects, and clinical considerations.
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
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
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
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2 Case Reports of Gastric Ultrasound
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
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
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.
2. Course Contents:
Introduction to Health biotechnology
Social acceptance of medical biotechnology
The molecular basis of disease,
Molecular and genetic markers
Detection of infectious agents
Active and passive immunization
vaccines ,Organ transplantation,
Applications of transgenic animals
Drug delivery systems, Blood transfusion,
Grafting techniques, Pharmacogenetics,
Strategies of gene therapy, gene delivery vehicles,
Biopharmaceuticals from plants
Uses of stem cell technology
3. Reference Books
“Medical Biotechnology” by Judit Pongracz, Mary
Keen “(2009). Elsevier Health Sciences.
“Biotechnology and Your Health: Pharmaceutical
Applications” by Bernice Zeldin Schacter, Bernice
Schacter (2005) Chelsea House Publishers,
“Health and Pharmaceutical Biotechnology” by D.M.
Chetan, K.P. Dinesh, D.M. Chetan (2006) Firewall
Media.
4. Introduction to health biotechnology
Applications
Drug production
Pharmacogenomics
Gene therapy
Genetic testing
OUTLINE (Lecture-I)
5. What Is Biotechnology?
Scientific processes to get new organisms
or new products from organisms.
It is the use of living organisms or processes to
develop products useful for mankind.
6. Has been existing since centuries
Begin with the first action of human on life for his
welfare
Term coined by a Hungarian engineer Karl Ereky
Modern biotechnology started in California in
1970’s
History
7. Origins of Biotechnology
Although it seems like a new thing,
biotechnology has actually been
around for a while:
Domesticated plants and animals are
the result of selective breeding
Using yeast to make bread rise
Using bacteria or yeast to ferment
grapes into wine
8. Any technique that uses living organisms
or substances from those organisms to
make or modify a product, to improve
plants or animals or to develop
microorganisms for specific uses
Definition
10. The use of biological methods to optimize industrial
processes
Applied by manufacturers of laundry detergents
Includes research for new enzymes (proteins that
remove oily and protein-based stains)
Enzymes that work under extreme conditions (wash
temperatures of 20°C or 90°C)
This often entails modifying the enzymes of
microorganisms for these processes
White biotechnology
11. Use of biotechnological techniques in agriculture
Vitamin A deficiency is a serious problem and can
cause blindness at a young age if left untreated
Golden rice was genetically modified to produce
beta-carotene (a precursor of vitamin A that the
body converts to vitamin A). A diet including
golden rice can thus help to raise vitamin A levels
Green biotechnology:
12. Also called red biotechnology
It includes:
o Production of medicines and pharmaceutical products for
treating or diagnosing disorders
o Designing of organisms to manufacture antibiotics and
vaccines
o Engineering of genetic defects through genomic
manipulation
o Use in forensics through DNA profiling
Biotechnology and medicine:
13. Production of human insulin from non- human sources.
Production of hormones like Interferons, Cytokinins,
Steroids and human growth hormones.
Gene therapy for prevention and control of diseases like
hemophilia cystic fibrosis
Development of vaccines and antibodies for rabies, HIV,
etc.
Examples…
14. Drug production
Pharmacogenomics
Gene therapy
Genetic testing
Health Biotechnology
15. It is the process in which pharmaceutical products
are produced through application of
biotechnological techniques
Medicines are produced for:
• Diagnosis
• Cure treatments
• Prevention of diseases
Drug production
16. Producing medicines through:
Isolating enzymes
Genetically engineering enzymes
Drug production
17. Recently, plants are being genetically modified to
produce pharmaceutical products instead of their
natural compounds
For Example:
A drug Elelyso for treating Gaucher is being
produced by genetically engineering carrots
Drug production
18. INSULIN:
Human insulin is being produced using
genetic engineering technique known as
humulin and it is used for the treatment of
diabetes that is low sugar level in the
blood…..
Drug production
19. INTERFERON:
o Interferon interfere in transmission of viral genome from
one cell to another and it also inhibits the cell division
of abnormal cells.
o Interferon produced using the recombinant DNA
technology is used to treat cancer patients.
o Interferon improved the quality of life of cancer
patients…..
Drug production
20. HUMAN GROWTH HORMONE:
Since dwarfism is caused by growth hormone
deficiency so it can be diagnose by HGH testing.
So HGH is used for the treatment of dwarfism due to
hypo pituitary activity.
Drug production
21. Pharma = Drug or Medicine
Genomics = The study of genes
Studying response of genetic make up of
an individual to a drug or pharmaceutical
products
Pharmacogenomics
22. “One-size-fits-all drugs” only work for about 60
percent of the population at best. And the other 40
percent of the population increase their risks
of adverse drug reaction because their genes do
not do what is intended of them.
Use of Pharmacogenomics:
23. Helps in the development of tailor made medicines
Ensures more appropriate methods of
determining drug dosages
Improve process of drug discovery and approval
Obtaining of better and safer vaccination
Decrease in the overall cost of Health Care
Advanced Screening for Disease
Impotance Of Pharmacogenomics
24.
25. Opinion:
This sort of card would initially (~2025) include
mostly information related to drug metabolizing
enzymes.
Around ~2050 it might include an entire individual
genome
Pharmacogenomics
SMART CARD
(Confidential)
26. Some barriers faced are:
Complexity of finding gene variation that affect
drug response
Limited drug alternatives
Disincentives for drug companies to make
multiple pharmacogenomic products
Educating healthcare providers
Pharmacogenomics
27. The process in which a faulty gene is
removed or replaced with its healthy copy to
restore the normal function of that gene
Gene therapy
28. Replacing a mutated gene that causes
disease with a healthy copy of the gene
Inactivating or “knocking out” a mutated gene
that is functioning improperly
Introducing the new gene that help fight a
disease
Gene therapy
29. Some common ways are:
Using fat droplets in nose sprays
Using cold viruses that are modified to carry
alleles ,go into the cell and affect them
The direct injection of DNA(might include
electroporation or biolistic method)
Gene therapy
30. The process of gene therapy is of two types:
Stem cell gene therapy:
In this gene therapy is applied on a fully developed
organism and the effects of gene therapy lasts only to
the operated organism
Germ line gene therapy:
In this process gene therapy is done on a fertilized egg
or an early embryo and the altered genome is followed in
next generations.
Gene therapy
32. 4) Tissue Engineering
A form of regenerative
medicine, tissue
engineering is the creation
of human tissue outside
the body for later
replacement.
Usually occurs on a tissue
scaffold, but can be grown
on/in other organisms as
shown on the right.
33. 4) Tissue Engineering
Tissue engineers have
created artificial skin,
cartilage and bone marrow.
Current projects being
undertaken include creating
an artificial liver, pancreas
and bladder.
Again, we are far from
replacing a whole organ, but
just looking for “refurbishing”
our slightly used ones at the
moment.
34. The examination of a patient’s DNA molecule
to determine his/her DNA sequence for
mutated genes
The genome of an individual is scaned for this
purpose by a scientist
Genetic testing
36. Better drugs can be obtained by the knowledge of
genetics
Genetic testing can be used to detect the
mutations regarding genetic disorders like cystic
fibrosis, sickle cell anaemia, hutington diseases,
etc.
Tests are also being developed to detect various
cancers
Genetic testing
38. Mutations Detection
Detection of mutations has central role in various areas
of genetic diagnosis
Preimplantation genetic diagnosis (PGD),
Prenatal diagnosis (PND)
Presymptomatic testing
Confirmational diagnosis
Forensic/identity testing.
Two groups of tests, molecular and cytogenetic, are
used in genetic syndromes.
39. Single Base Pair Mutations
Direct sequencing, DNA hybridization and/or
restriction enzyme digestion methods are used for
detection of single pair mutations. However, there
are two approaches for genetic diagnosis;
Indirect approach depends on the results from a
genetic linkage analysis using DNA markers such as
STR(short tandem repeat) or VNTR (variable number
tandem repeat) markers flanking or within the gene
Direct approach for diagnosis essentially depends on
the detection of the genetic variations responsible for
the disease
40. Cytogenetics and
Molecular Diagnostics
Karyotyping
Fluorescence in situ hybridization (FISH)
Comparative genomic hybridization (CGH)
Molecular Diagnostics
(Known & Unknown Mutations)
Next Generation Sequencing (NGS)
41. Karyotyping
Karyotyping is the process of pairing and ordering all
the chromosomes of an organism, thus providing a
genome-wide image of an individuals chromosomes
Karyotypes are prepared from mitotic cells which are frozen
in metaphase.
Characteristic structural features for each chromosome are
revealed.
Can reveal changes in chromosome numbers linked to
conditions such as Down’s syndrome.
Careful analysis can show more subtle changes as
chromosomal deletions, duplications, translocations or
inversions.
There is an increasing use of karyotyping for diagnosis of
specific birth defects and genetic disorders.
42. Karyotyping Applications
Chromosome studies are advised in the following
situations:
Suspected chromosome abnormality
Sexual disorders
Multiple congenital anomalies/ developmental
retardation
undiagnosed learning disabilities
Infertility or multiple miscarriage
Stillbirth and malignancies
43. Preparation of visual karyotype
Traditionally, the microscopic study
of chromosomes is performed on
compacted chromosomes at a
magnification of 1000 at metaphase.
Cells are arrested at metaphase
stage with a microtubule
polymerization inhibitor such as
colchicine
These cells are spread on a glass
slide and stained with Giemsa stain
(G banding).
Chromosomes are studied by
making a photograph or digital
imaging and subsequent assembling
of chromosomes
45. Human Karyotype
Human chromosomes are categorized
based on position of centromere;
Metacentric; the centromere at center (chromosomes
1, 3, 16, 19 and 20),
Acrocentric; the centromere near one end
(chromosomes 13, 14, 15, 21, 22 and Y are)
other chromosomes are submetacentric
The convenient methods of chromosome banding are
G-(Giemsa), R-(reverse),C-(centromere) and Q-
(quinacrine) banding
47. Fluorescence in situ
hybridization (FISH):
FISH is applied to provide specific localization of genes on
chromosomes.
Rapid diagnosis of trisomies and microdeletions is
acquired using specific probes.
Usually a denatured probe is added to a metaphase
chromosome spread and incubated overnight to allow
sequence-specific hybridization.
After washing off the unbound probe, the bound probe is
visualized by its fluorescence under UV light; thus, the site
of the gene of interest is observed as in situ
50. Comparative genomic
hybridization (CGH)
CGH, a special FISH technique (dual probes), is applied
for detecting all genomic imbalances.
The basics of technique is comparison of total genomic
DNA of the given sample DNA (e.g. tumor DNA) with total
genomic DNA of normal cells.
Typically, an identical amount of both tumor and normal
DNAs is labeled with two different fluorescent dyes; the
mixture is added and hybridized to a normal lymphocyte
metaphase slide.
A fluorescent microscope equipped with a camera and an
image analysis system are used for evaluation
Copy number of genetic material (gains and losses) is
calculated by evaluation software.
51. CGH is used to determine copy
number alterations of genome in
cancer and those cells whose
karyotype is hard or impossible
to prepare or analyze.
In array-CGH, metaphase slide is
replaced by specific DNA
sequences, spotted in arrays on
glass slides, so its resolution is
increased.
Comparative genomic
hybridization (CGH)
53. Molecular Diagnostics
Molecular methods for identification of the disease-
causing mutations could be classified as methods for
known and methods for unknown mutations.
Several criteria, have to be met for choosing a suitable
method; for example
type of nucleic acid (DNA or RNA)
kind of specimen (blood, tissues, etc.)
the number of mutations
reliability of the method
54. Detection of Known Mutations
Many different approaches have been used for
identifying known mutations
Polymerase chain reaction (PCR) and its versions
DNA microarray
DNA Sequencing
Multiplex ligation-dependent probe amplification
(MLPA)
55. Detection of Unknown Mutations
Single Strand Conformational
Polymorphism (SSCP)
Denaturing Gradient Gel Electrophoresis
(DGGE)
Restriction fragment length polymorphism
(RFLP)
56. 1. Polymerase chain reaction
In 1980s, Dr Mullis introduced a method for
amplifying DNA fragment to a large number of
fragments by polymerase chain reaction (PCR)
Essential components of PCR are template DNA,
primers , thermostable DNA polymerase enzyme
(e.g. Taq), divalent cations (usually Mg2+),
deoxynucleoside triphosphates (dNTPs) and
buffer solution
PCR, consisting of 25-40 repeated cycles, has
three discrete steps of temperature changes
57. Steps of PCR
Initial denaturation step includes heating the reaction to a temperature
of 92–96°C for 1–9 minutes.
1) Denaturation step includes heating the reaction to 92–98°C for 20–
30 seconds. The hydrogen bonds between complementary bases are
disrupted and DNA molecules are denatured, yielding single-stranded
DNA molecules (DNA melting).
2) Annealing step is performed by decreasing temperature to 50–
65°C for 25–40 seconds; so the primers are annealed to their targets
on single stranded DNAs by hydrogen bonds and a polymerase can
bind to the primer-template hybrid and begin DNA polymerization in
next step.
3) Extension step includes polymerization of the bases to the primers;
a thermostable such as Taq polymerase extends a new strand
complementary to the DNA template strand by adding matched dNTPs
in 5' to 3' direction at a temperature of 72°C.
A series of 25-40 repeated cycles of denaturation, annealing of
primers and extension is performed to amplify the template fragment.
Subsequently, a final elongation is sometimes done at 70–74°C for 5–
15 minutes after the last PCR cycle to ensure full extension of any
remaining single-stranded DNA
58. Types and Applications of PCR
1) Reverse transcriptase PCR (RT-PCR)
2) Multiplex PCR
3) Nested PCR
4) Amplification refractory mutation system
(ARMS) PCR:
5) Real time PCR
59. 1. Reverse transcriptase PCR
(RT-PCR)
In this version, a strand of RNA molecule is
transcribed reversely into its complementary
DNA (cDNA) using the reverse transcriptase
enzyme.
This cDNA is then amplified by PCR.
RT-PCR is applied to study the mutations at
RNA level.
60. 2) Multiplex PCR:
In this technique, multiple selected target regions in
a sample are amplified simultaneously using
different pairs of primers.
3) Nested PCR:
It includes two successive PCRs;
the product of the first PCR reaction is used as a
template for the second PCR.
This type of PCR is employed to amplify templates
in low copy numbers in specimens.
It has the benefits of increased sensitivity and
specificity.
61. 4) Amplification refractory
mutation system (ARMS) PCR:
Allele-specific amplification (AS-PCR) or ARMS-PCR is a general
technique for the detection of any point mutation or small
deletion
The genotype (normal, heterozygous and homozygous states) of a
sample could be determined using two complementary reactions:
one containing a specific primer for the amplification of normal DNA
sequence at a given
locus and the other one containing a mutants pecific primer for
amplification of mutant DNA.
ARMS-PCR has been used to check the most common mutation
in GJB2 gene, 35delG mutation
among deaf children.
5) Real time PCR:
In this technique, the amplified DNA is detected as the PCR
progresses.
It is commonly used in gene expression studies and quantification
of initial copy number of the target
62. DNA microarray
DNA “chips” or microarrays have
been used as a possible testing
for multiple mutations
Single DNA strands including
sequences of different targets
are fixed to a solid support in an
array format.
On the other hand, the sample
DNA or cDNA labeled with
fluorescent dyes is hybridized to
the chip
Then using a laser system, the
presence of fluorescence is
checked; the sequences and
their quantities in the sample are
determined
63. DNA Sequencing The main aim of DNA sequencing is to
determine the sequence of small regions of
interest (~1 kilobase) using a PCR product as
a template.
Dideoxynucleotide sequencing or Sanger
sequencing represents the most widely used
technique for sequencing DNA
In this method, double stranded DNA is
denatured into single stranded DNA with
NaOH
A Sanger reaction consists of a single strand
DNA, primer, a mixture of a particular ddNTP
with normal dNTPs (e.g. ddATP with dATP,
dCTP, dGTP, and dTTP).
A fluorescent dye molecule is covalently
attached to the dideoxynucleotide. ddNTPs
cannot form a phosphodiester bond with the
next deoxynucleotide so that they terminate
DNA chain elongation.
This step is done in four separate reactions
using a different ddNTP for each reaction
DNA sequencing could be used to check all
small known and unknown DNA variations.
64. Multiplex ligation-dependent
probe amplification (MLPA)
MLPA is commonly applied to screen deletions and
duplications of up to 50 different genomic DNA or
RNA sequences.
Altogether gene deletions and duplications account up to
10%, and in many disorders up to 30% of disease-causing
mutations
The probe set is hybridized to genomic DNA in solution
Each probe consists of two halves; one half is composed
of a target specific sequence and a universal primer
sequence, and other half has other more sequences, a
variable length random fragment to provide the size
differences for electrophoretic resolution.
65. Multiplex ligation-dependent
probe amplification (MLPA)
A pair of probes is hybridized on the target region
adjacently so that they can then be joined by use of a
ligase; the contiguous probe can be amplified by
PCR
After PCR amplification, the copy number of target
sequence i.e. deletion or duplication of target
sequence can be determined and quantified using
the relative peak heights
67. Detection of Unknown Mutations
Single Strand Conformational Polymorphism (SSCP)
Denaturing Gradient Gel Electrophoresis (DGGE)
Heteroduplex analysis
Restriction fragment length polymorphism (RFLP)
68. Single Strand Conformational
Polymorphism (SSCP)
SSCP is one of the simplest screening
techniques for detecting unknown
mutations (microlesions) such as
unknown single-base substitutions,
small deletions, small insertions, or
micro-inversions
A DNA variation causes alterations in
the conformation of denatured DNA
fragments during migration within gel
electrophoresis
The logic is comparison of the altered
migration of denatured wild-type and
mutant fragments during gel
electrophoresis
69. Single Strand Conformational
Polymorphism (SSCP)
DNA fragments are denatured, and renatured under special
conditions preventing the formation of double-stranded
DNA and allowing conformational structures to form in
single-stranded fragment
The conformation is unique and resulted from the primary
nucleotide sequence
Mobility of these fragments is differed through non-
denaturing polyacrylamide gels; detection of variations is
based on these conformational structures.
PCR is used to amplify the fragments, called PCR-SSCP,
because the optimal fragment size can be 150 to 200 bp.
About 80–90% of potential point mutations are
detected by SSCP
70. Denaturing Gradient Gel
Electrophoresis (DGGE):
DGGE has been used for screening of
unknown point mutations. It is based on
differences in the melting behavior of
small DNA fragments (200-700 bp);
even a single base substitution can
cause such a difference.
In this technique, DNA is first extracted
and subjected to denaturing gradient gel
electrophoresis.
As the denaturing condition increases,
the fragment completely melts to single
strands.
The rate of mobility in acrylamide gels
depends on the physical shape of the
fragment
71. Denaturing Gradient Gel
Electrophoresis (DGGE):
Detection of mutated fragments would be possible by
comparing the melting behavior of DNA fragments on
denaturing gradient gels.
Approximately less than 100% of point mutations can
be detected using DGGE.
Maximum of a nearly 1000 bp fragment can be
investigated by this technique
72. Heteroduplex analysis
A mixture of wild-type and mutant DNA molecules is
denatured and renatured to produce heteroduplices
Homoduplices and heteroduplices show different
electrophoretic mobilities through nondenaturing
polyacrylamide gels
In this technique, fragment size ranges between 200
and 600 bp, Nearly 80% of point mutations have
been estimated to be detected by heteroduplex
analysis
73. Restriction fragment length
polymorphism (RFLP)
Point mutations can change
restriction sites in DNA causing
alteration in cleavage by
restriction endonucleases which
produce fragments with various
sizes
RFLP is used to detect
mutations occurring in restriction
sites
75. Next Generation Sequencing
High speed and throughput, both qualitative and quantitative
sequence data are allowed by means of NGS technologies so
that genome sequencing projects can be completed in a few days
NGS systems provide several sequencing approaches including
whole-genome sequencing (WGS), whole exome sequencing
(WES), transcriptome sequencing, methylome, etc.
The coding sequences compromises about 1% (30Mb) of the
genome.
More than 95% of the exons are covered by WES; on the other
hand, 85% of disease-causing mutations in Mendelian disorders
are located in coding regions. Sequencing of the complete coding
regions (exome), therefore, could potentially uncover the
mutations causing rare, mostly monogenic, genetic disorders as
well as predisposing variants in common diseases and cancer.