Medical

Biotechnology
Introduction
Detecting and Diagnosing

Human Diseases Conditions

 Introduction
Applications of medical biotechnology have
existed for decades, for instance :
100 years ago : leeches used to treat ilness
(especially blood diseases) by bloodletting
Now : enzymes found in its saliva that can
dissolve blood clots and used to treat strokes
and heart attacks
 The Power of Medical
Biotechnology
Detecting and Diagnosing Human Disease
Conditions
Medical Products and Applications of
Biotechnology
Gene Therapy

Regenerative Medicine

Human Genome Project

Detecting and Diagnosing
Human Disease Conditions
Models of Human Diseases
Detecting Genetics Diseases
 Models of Human Disease

Human genome project is to develop a better

understanding of genetics similarities and
differences between humans and other species
We share 50% of our genes with fruit flies, 40%
with roundworms, 31% with yeast and 90% with
mice
A number of human genetic diseases also
occurs in model organisms : mice, rats, chicks,
yeast, fruit flies, worms, frogs and zebrafish
Approximately 61% of gene mutated in 289
human disease conditions (includes prostate
cancer, pancreatic cancer, cystic fibrosis,
leukemia, etc) are found in fruit flies.
 Models of Human Disease

We can use these model organisms to identify

disease genes and test gene therapy
approaches to check their effectiveness and
safety before using them to clinicals trials in
humans
Homologues : genes that have been identified in
models species have been shown to be related
to human genes based on DNA sequence
similarity
A gene can be eliminated in model organisms
through gene knockout
 Models of Human Disease,
for instance :
Mice can become obese, if they lack ob gene, codes for
leptin (a hormone), which produced by fat cells and
travels through bloodstream to the brain to regulate
hunger providing insight on fat metabolism in humans
and the genetics that may influence weight disorders
Apoptosis, a programmed cell death, have been greatly
advanced by studies of the roundworms Caenorhabditis
elegans. Apoptosis is responsible for :
- the degeneration of webs between the fingers and toes
- neurodegenerative diseases, such as Alzheimer,
Hutington, Lou Gehrig and Parkinsons disease, as
well as arthritis and forms of infertility
 Models of Human Disease,
for instance :
Model organisms will help us understand the
genes involved and slow or stop these
degenerative process
Models of Human Disease,
for instance :
Using gene knockouts to develop heart attack
mice that are deficient in genes required for
cholesteral metabolism show elevated
blood cholesterol similar to atherosclerosis
they can test therapy to combat heart disease
Creating a rodent model for AIDS, but HIV
does not recognize and bind to receptor on
mouse T cells express human T cell
proteins in mice to trick the virus into infecting
mice
 Detecting Genetics Diseases
Testing for chromosome abnormalities and
defective genes
Single nucleotide polymorphisms

Identifying sets of disease genes by

microarrays analysis
Testing for chromosome
abnormalities and defective genes
Most genetic testing occurred on fetuses for
identifying the sex of a child or to detect a
small number of genetic disease occur as
a result of alterations in chromosome number
problem : separation during the formation
of sperm or eggs cells
 Abnormal Chromosome Number

When nondisjunction Meiosis I

occurs
Pairs of homologous
chromosomes do not Nondisjunction

separate normally Meiosis II

during meiosis
Gametes contain two
Nondisjunction
copies or no copies Gametes

of a particular
chromosome
n+1 n+1 n1 n1 n+1 n 1 n n
Number of chromosomes

(a) (b)
Nondisjunction of homologous Nondisjunction of sister
chromosomes in meiosis I chromatids in meiosis II
Abnormal Chromosome Number

Example : Down syndrome have 3 copies

of chromosomes 21 (Trisomy 21), related to
the age of eggs produced by the female
Testing for chromosome
abnormalities and defective genes
Two different methods can be used to test a
developing fetus :
Amniocentensis
Chrionic villus sampling

Another techniques for karyotyping in both fetuses

and adults (from white blood cells) :
Fluorescence in situ hybridization (FISH)
Restriction fragment length polymorphism (RFLP)
analysis
Allele specific oligonucleotide (ASO) analysis
Amniocentensis
Amniocentensis is performed when the fetus is
around 16 weeks of age
A needle is inserted into the pocket of amniotic fluid
(contains cells shed from the fetus) cultured for
few days to increase cell number karyotype : the
cells are treated to release their chromosomes
which are spread onto a glass slide stained with
different dyes and creating patterns of light and dark
bands on each chromosome.
Based on the size and banding pattern of
chromosomes pairs and count chromosome
number
 Human
chromosomes

male

female
Chorionic villus sampling
A suction tube is used to remove a chrionic villi
(fetal tissue that helps from the placenta)
Advantage :
- enough cells are obtained can immediatelly
used for karyotyping
- this procedure can be done around 8-10 weeks
Disadvantage :
- this procedure carrier a higher risk for disturbing
the fetus and causing a miscarriage
Flourescence in situ
hybridization (FISH)
Chromosomes are spread on a slide and
then fluorescent probes are hybridized with
complementary sequences to each
chromosome.
If the probes that fluoresce different colors
produces a spectral karyotype
FISH can be used to :

- identifying missing or extra chromosomes

- to detect defective chromosome
Flourescence in situ
hybridization (FISH)
Several human genetic disease created by
chromosomal abnormalities occur when a
portion of a chromosome is swapped from one
chromosome to another because of problems in
chromosome replication
Chronic myelogenous leukemia : DNA
exchanged between chromosomes 9 and 22
can be detected by FISH
Chronic myelogenous leukemia

Arecaused by translocations of
chromosomes

Normal chromosome 9 Translocated chromosome 9

Reciprocal
translocation

Philadelphia
chromosome

Normal chromosome 22 Translocated chromosome 22

Restriction fragment length
polymorphism (RFLP) analysis
RFLP can detect individual diseased genes
result from mutations
Basic idea : defective gene sequences may
be cut differently by restriction enzymes than
their normal complements, because
nucleotide changes can affect restriction
enzymes cutting sites create more or
fewer cutting sites
These DNA fragments are subjected to
Southern blot analysis with a specific probe
RFLP
Restriction Fragment Length Polymorphism
differences in DNA between individuals

change in DNA sequence affects

restriction enzyme cut site
will create different band pattern
RFLP Process
Restriction fragment length
polymorphism (RFLP) analysis
Disadvantage : it can only be used to analyze
gene defects in which mutation changes a
restriction enzymes cutting sites developed
by ASO analysis
Allele specific oligonucleotide
(ASO) analysis
DNA is isolated from white blood cells, amplified by
PCR using primers that flank a disease gene
blotted onto nylon filters and hybridized separately
to two different ASOs probes (small ssDNA around
20 bp).
An ASO for normal gene will hybridize to a normal
gene and an ASO for mutant gene will hybridize to a
mutant gene
PCR and ASO analysis can be used to detect gene
defects in single cells from early embryos
Single nucleotide
polymorphisms (SNPs)
SNPs are single nucleotide changes or
mutations in DNA sequences that vary
from individual to individual
Approximately 99.9% of the DNA
sequence from different people will be
exactly the same. About 80% of the 0.1%
variation will be SNPs.
One SNP occurs every 1,000-3,000 bp.
Over 1.4 million SNPs have been identified
to date.
SNP Variations in The Human
Genome

Mechanisms of Aging and Development (2003);124: 17-25.

Single nucleotide
polymorphisms (SNPs)
Most SNPs have no effect on a cell because
they occur in non-protein coding regions
(intron).
When a SNP occurs in a gene sequence
change in protein structure that produces
disease or influences traits
Single nucleotide
polymorphisms (SNPs)
SNPs represent variations in DNA sequence
that influence how we respond to stress and
disease different forms of disease
Some SNPs might be used to predict
susceptibilities to diseases such as arthitis,
stroke, cancer, heart disease, diabetes,
behavioral and emotional illness
Gene Microarrays
DNA microarrays is a technique to screen a patient
for pattern of genes that might be expressed in a
particular disease condition.
A microarray (DNA chip) is created using a small
glass microscope slide.
A ssDNA are attached/spotted onto the slides using
an arrayer (a computer-controlled high-speed
robotic arm), fitted with a number of tiny pins.
Each pin is immersed in a small amount of solution
of different DNA molecules (such as cDNAs from
different genes)
Gene Microarray

Prehybridization Posthybridization

Fluorescence
labeled
target DNA

Synthetic DNA probes Probes with

hybridized DNA
 Gene Microarrays

slide with spots of DNA

each spot = 1 gene

Create a slide with a sample of each gene from the

organism
each spot is one gene
Convert mRNA labeled cDNA mRNA cDNA
mRNA from cells

reverse
transcriptase
 Gene Microarrays

slide with spots of DNA

each spot = 1 gene

Labeled cDNA hybridizes with DNA on slide

each yellow spot = gene matched to mRNA

each yellow spot = expressed gene

mRNA cDNA cDNA matched to genomic DNA

Gene Microarrays

2-color fluorescent tagging

Comparing treatments or conditions =

Measuring change in gene expression
sick vs. healthy; cancer vs. normal cells
before vs. after treatment with drug
different stages in development
Gene Microarrays

2-color fluorescent tagging

Color coding: label each condition with different

color
red = gene expression in one sample
green = gene expression in other sample
yellow = gene expression in both samples
black = no or low expression in both
Treatment strategies are being designed base on
these differences
Protein microarrays
Protein microarrays are used in the same
way that DNA microarrays are used.
These chips contain hundreds/thousands of
antibodies spotted on a chip
Blood protein applied to the chip detect
illness indicated by the presence of proteins
from disease-causing organism.