Health biotechnology

HEALTH

BIOTECHNOLOGY

Rizwan Abbas baho

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

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.

Introduction to health biotechnology

Applications

Drug production

Pharmacogenomics

Gene therapy

Genetic testing

OUTLINE (Lecture-I)

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.

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

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

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

Green biotechnology (agricultural)

Red biotechnology (medical)

Blue biotechnology (aquatic)

White biotechnology (industrial)

Applications

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

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:

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:

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…

Drug production

Pharmacogenomics

Gene therapy

Genetic testing

Health Biotechnology

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

Producing medicines through:

Isolating enzymes

Genetically engineering enzymes

Drug production

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

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

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

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

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

“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:

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

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)

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

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

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

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

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

Gene therapy

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.

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.

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

Forensic/identity testing

Determining sex

Conformational diagnosis of symptomatic

individuals

Newborn screening

Prenatal diagnostic screening

Genetic testing

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

Mutation

Detection

Dr. Sajjad Ahmad

Mutations DetectionDetection 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.

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

Cytogenetics and

Molecular Diagnostics

Karyotyping

Fluorescence in situ hybridization (FISH)

Comparative genomic hybridization (CGH)


(Known & Unknown Mutations)

Next Generation Sequencing (NGS)

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.

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

Preparation of visual karyotype Traditionally, the microscopic study

of chromosomes is performed oncompacted chromosomes at amagnification of 1000 at metaphase.

Cells are arrested at metaphasestage with a microtubulepolymerization inhibitor such ascolchicine

These cells are spread on a glassslide and stained with Giemsa stain(G banding).

Chromosomes are studied bymaking a photograph or digitalimaging and subsequent assemblingof chromosomes

Process of Karyotyping

Human KaryotypeHuman 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

Fluorescence in situ

hybridization (FISH)

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

Comparative genomic

hybridization (CGH)

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 genomicDNA of the given sample DNA (e.g. tumor DNA) with totalgenomic DNA of normal cells.

Typically, an identical amount of both tumor and normalDNAs is labeled with two different fluorescent dyes; themixture is added and hybridized to a normal lymphocytemetaphase slide.

A fluorescent microscope equipped with a camera and animage analysis system are used for evaluation

Copy number of genetic material (gains and losses) iscalculated by evaluation software.

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)


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

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)

Detection of Unknown Mutations

Single Strand Conformational

Polymorphism (SSCP)

Denaturing Gradient Gel Electrophoresis

(DGGE)

Restriction fragment length polymorphism

(RFLP)

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

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 aredisrupted and DNA molecules are denatured, yielding single-strandedDNA 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 targetson single stranded DNAs by hydrogen bonds and a polymerase canbind to the primer-template hybrid and begin DNA polymerization innext step.

3) Extension step includes polymerization of the bases to the primers;a thermostable such as Taq polymerase extends a new strandcomplementary to the DNA template strand by adding matched dNTPsin 5' to 3' direction at a temperature of 72°C.

A series of 25-40 repeated cycles of denaturation, annealing ofprimers 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 anyremaining single-stranded DNA

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

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.

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.

4) Amplification refractory mutation system (ARMS) PCR: Allele-specific amplification (AS-PCR) or ARMS-PCR is a generaltechnique for the detection of any point mutation or smalldeletion

The genotype (normal, heterozygous and homozygous states) of asample could be determined using two complementary reactions:

one containing a specific primer for the amplification of normal DNAsequence at a given

locus and the other one containing a mutants pecific primer foramplification of mutant DNA.

ARMS-PCR has been used to check the most common mutationin GJB2 gene, 35delG mutation

among deaf children.

5) Real time PCR:In this technique, the amplified DNA is detected as the PCRprogresses.

It is commonly used in gene expression studies and quantificationof initial copy number of the target

DNA microarray DNA “chips” or microarrays have

been used as a possible testingfor multiple mutations

Single DNA strands includingsequences of different targetsare fixed to a solid support in anarray format.

On the other hand, the sampleDNA or cDNA labeled withfluorescent dyes is hybridized tothe chip

Then using a laser system, thepresence of fluorescence ischecked; the sequences andtheir quantities in the sample aredetermined

DNA Sequencing The main aim of DNA sequencing is to

determine the sequence of small regions ofinterest (~1 kilobase) using a PCR product asa template.

Dideoxynucleotide sequencing or Sangersequencing represents the most widely usedtechnique for sequencing DNA

In this method, double stranded DNA isdenatured into single stranded DNA withNaOH

A Sanger reaction consists of a single strandDNA, primer, a mixture of a particular ddNTPwith normal dNTPs (e.g. ddATP with dATP,dCTP, dGTP, and dTTP).

A fluorescent dye molecule is covalentlyattached to the dideoxynucleotide. ddNTPscannot form a phosphodiester bond with thenext deoxynucleotide so that they terminateDNA chain elongation.

This step is done in four separate reactionsusing a different ddNTP for each reaction

DNA sequencing could be used to check allsmall known and unknown DNA variations.

Multiplex ligation-dependent

probe amplification (MLPA)

MLPA is commonly applied to screen deletions andduplications of up to 50 different genomic DNA orRNA sequences.

Altogether gene deletions and duplications account up to10%, and in many disorders up to 30% of disease-causingmutations

The probe set is hybridized to genomic DNA in solution

Each probe consists of two halves; one half is composedof a target specific sequence and a universal primersequence, and other half has other more sequences, avariable length random fragment to provide the sizedifferences for electrophoretic resolution.



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

Detection of Unknown Mutations

Single Strand Conformational Polymorphism (SSCP)

Denaturing Gradient Gel Electrophoresis (DGGE)

Heteroduplex analysis

Restriction fragment length polymorphism (RFLP)


Polymorphism (SSCP)

SSCP is one of the simplest screeningtechniques for detecting unknownmutations (microlesions) such asunknown single-base substitutions,small deletions, small insertions, ormicro-inversions

A DNA variation causes alterations inthe conformation of denatured DNAfragments during migration within gelelectrophoresis

The logic is comparison of the alteredmigration of denatured wild-type andmutant fragments during gelelectrophoresis


Polymorphism (SSCP) DNA fragments are denatured, and renatured under special

conditions preventing the formation of double-strandedDNA and allowing conformational structures to form insingle-stranded fragment

The conformation is unique and resulted from the primarynucleotide sequence

Mobility of these fragments is differed through non-denaturing polyacrylamide gels; detection of variations isbased 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 aredetected by SSCP

Denaturing Gradient Gel

Electrophoresis (DGGE): DGGE has been used for screening of

unknown point mutations. It is based ondifferences in the melting behavior ofsmall DNA fragments (200-700 bp);even a single base substitution cancause such a difference.

In this technique, DNA is first extractedand subjected to denaturing gradient gelelectrophoresis.

As the denaturing condition increases,the fragment completely melts to singlestrands.

The rate of mobility in acrylamide gelsdepends on the physical shape of thefragment

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

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

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

Next Generation Sequencing

Next Generation Sequencing

High speed and throughput, both qualitative and quantitativesequence data are allowed by means of NGS technologies sothat genome sequencing projects can be completed in a few days

NGS systems provide several sequencing approaches includingwhole-genome sequencing (WGS), whole exome sequencing(WES), transcriptome sequencing, methylome, etc.

The coding sequences compromises about 1% (30Mb) of thegenome.

More than 95% of the exons are covered by WES; on the otherhand, 85% of disease-causing mutations in Mendelian disordersare located in coding regions. Sequencing of the complete codingregions (exome), therefore, could potentially uncover themutations causing rare, mostly monogenic, genetic disorders aswell as predisposing variants in common diseases and cancer.

Health biotechnology

Health & Medicine

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