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.

1. HEALTH
BIOTECHNOLOGY
Rizwan Abbas baho

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

9. Green biotechnology (agricultural)
Red biotechnology (medical)
Blue biotechnology (aquatic)
White biotechnology (industrial)
Applications

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

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

31. 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

35.  Forensic/identity testing
 Determining sex
 Conformational diagnosis of symptomatic
individuals
 Newborn screening
 Prenatal diagnostic screening
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

37. Mutation
Detection
Dr. Sajjad Ahmad

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

44. Process of Karyotyping

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

46. Fluorescence in situ
hybridization (FISH)

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

49. Comparative genomic
hybridization (CGH)

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)

52. Molecular Diagnostics

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

66. Multiplex ligation-dependent
probe amplification (MLPA)

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

74. Next Generation Sequencing

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.