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7/29/2019 DNA Diagnosis of Genetic Diseases
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Presented by
SharanabasappaIIst M.Pharm.Dept. of Pharmacology
Subject In ChargeDr. THIPPESWAMY B. S.
M.Pharm., Ph.DProfessor & Head of the Department
DEPT. OF PHARMACOLOGYSSCP, TUMKUR. 1SSCP, Tumkur
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CONTENTS Introduction
Genetic diseases
Methods of DNA Assay
Importance of DNA in the diagnosis of genetic
diseases
Some of the important genetic diseases for which
DNA analysis is used
Diagnostic centers in India
References 2SSCP, Tumkur
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INTRODUCTIONDiagnosis of diseases due to pathogens or due to
inherent genetic defects is necessary for appropriatetreatment.
Traditional diagnostic methods for parasite infectionsinclude microscopic examination, in vitro culture, &detection of antibodies in serum.
And for genetic diseases, the procedures such asestimation of metabolites (blood/urine) & enzyme assaysare used.
These laboratory techniques are indirect & not alwaysspecific.
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Continued ……
DNA, being the genetic material of the livingorganisms, contains the information which contributesto various characteristic features of the specificorganism.
Thus, the presence of a disease-causing pathogen can be detected by identifying a gene or a set of genes of the organism.
Inherited genetic defect can be diagnosed by identifying the alterations in the gene.
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GENETIC DISORDERS • DEFINITION:- A disease or disorder which is inherited
genetically.
• TYPES:- Five types
1) Chromosomal EX: Down syndrome
2) Single-gene (also called Mendelian or monogenic)EX: cystic fibrosis, sickle cell anemia
3) Cancer
4) Multi-factorial (also called complex or polygenic)EX: Alzheimer’s disease, arthritis, diabetes, cancer, and
obesity
5) Mitochondrial 5SSCP, Tumkur
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METHODS OF DNA ASSAY
1) Nucleic acid hybridization
a) radioactive detection system
b) non – radioactive detection system
2) DNA probes
a) PCR in the use of DNA probes
b) DNA probes & signal amplification
3) DNA chip – microarray of gene probe
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NUCLEIC ACID HYBRIDIZATION
• Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two ormore complementary strands of nucleic acids into asingle hybrid.
• There are two types of DNA hybridization techniques:
a) Radioactive detection system
b) Non-radioactive detection system
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Continued …… • PRINCIPLE:-Single stranded DNA molecule recognize and
specifically bind to a complementary DNA strand in a mixture
of other DNA strand. This is comparable to a specific key and
lock relationship.
• BASIC PROCEDURE:-
-Single stranded target DNA is bound to a membrane support
↓
-DNA probe labeled with detector substance is added↓
-DNA probe pairs with the complementary target DNA
↓ wash unbound DNA probes
-Sequence of nucleotide in the target DNA can be identified8SSCP, Tumkur
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Radioactive detection system
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Continued ……
The DNA probe tagged with a radioactive isotope (commonly phosphorus 32)
target DNA is purified & denatured
mixed with DNA probe
Isotope labeled DNA molecules specifically hybridizes with the target DNA
Presence of radioactivity in the hybridized DNA, detected by autoradiography.
Non – hybridized probe DNA is washed away
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Continued ……
• Disadvantages of Radioactive detection system
isotopes have short half life’s
risks in handling
requiring special laboratory equipments.
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Non-Radioactive detecting system
• Principle:- Detection is based on enzymatic conversion of a
chromogenic (colour producing) or chemiluminescent (light
emitting) substrates. Mainly Biotin-labeled (Biotinylated)
nucleotides are incorporated into DNA probe.
• Advantages:-
Biotin-labeled DNA is quite stable at RT for about 1 year.
Chemiluminescence detection is very sensitive thanchromogenic detection system
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Continued …..
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DNA PROBE/GENE PROBE
• Synthetic single stranded DNA molecule that can recognizeand specifically bind to a target DNA by complimentary base
pairing in a mixture of bio molecules.
• DNA probes are either long (>100 nucleotides) or short (<50
nucleotides)
• Bind to the total or a small portion of the target DNA.
• Most important requirement is their specific & stable binding
with target DNAs.
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Continued …… • MOA:
Basic principle (Hybridization of DNA) i.e. Denaturation &
Renaturation.
When a ds DNA molecule is subjected to physical or
chemical changes, the H-bonds break & complementary
stands get separated.
Under suitable conditions (i.e. temp., pH, salt conc.), the twoseparated single DNA strands can reassemble to form the
original ds DNA.
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Continued ……
• Methods used to obtain DNA probes Majority of DNA probes are chemically synthesized in the
laboratory.
Many other ways are:1) Isolation of selected regions of genes
2) Cloning of intact genes
3) Producing from mRNA
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Continued ……
• Isolation of selected regions of genes The DNA is cut by RENs.
The DNA fragment is cloned in vectors.
DNA probes are selected by screening.
• Synthesis of DNA probes from mRNA mRNA molecules specific to a particular DNA sequence are
isolated. By using Reverse Transcriptase cDNA molecules are
synthesized & used as a probe to detect the target DNA.
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PCR in the use of DNA probes
• Detection of target sequence becomes quite difficult if the
quantity of DNA is very low.
• Therefore, Polymerase Chain Reaction is first employed to
amplify the minute quantities of target DNA & identified by
a DNA probe.
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DNA probes & signal amplification
• It is an alternative to PCR for the identification of minute
quantities of DNA by using DNA probes.
• In PCR, target DNA is amplified, while in signal amplification,
the target DNA bound to DNA probe is amplified.
•Two general methods to achieve signal amplification.
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Continued ……
1) Separate the DNA target – DNA probe complex from the
rest of the DNA molecules, & then amplify it.
2) Amplify the DNA probe (bound to target DNA) by using a
second probe. The RNA complementary to the DNA probe
can serve as the second probe.
The RNA-DNA-DNA complex can be separated &lified. The O-beta replicase which catalyses RNA
replication is used.
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DNA Chip – Microarray of Gene Probes
• DNA chip or Genechip contains thousands of DNA probes
(4000,000 or even more) arranged on a small glass slide of the
size of a postage stamp.
• Thousands of target DNA molecules can be scannedsimultaneously.
Advantages:- Very rapid
Sensitive & Specific
Simultaneous analysis of many DNA is possible 22SSCP, Tumkur
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Continued ……
• TechniqueUnknown DNA molecules cut into fragments by RENs
↓
Fluorescent marker are attached to these DNA fragments
↓
Allowed to react with probes of the DNA chip
↓ Target DNA fragments with complementary sequences bind to DNA
probes
↓
Remaining DNA fragments are washed away
↓ Target DNA pieces can be identified by their fluorescence emission by
passing a laser beam
↓
Computer records the pattern of fluorescence emission & DNA
identification 23SSCP, Tumkur
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Continued ……
• Applications
Presence of mutations in a DNA sequence is identified.
Genechip probe array has been successfully used for thedetection of mutations in the p53 & BRCA 1 genes (involved in
cancer).
Scientists are trying to develop Genechips for the entire
genome of an organism.
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Importance of DNA in the Diagnosisof Genetic Diseases
Traditional laboratory tests for the diagnosis of genetic
diseases are mostly based on the estimation of metabolites
&/or enzymes.
Usually done after the onset of symptoms. DNA analysis can specifically diagnose the inherited disease
at the genetic level.
DNA based tests are useful to discover, well in advancewhether the individuals or their offsprings are at risk for
any genetic diseases.
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Continued ……
Also used for prenatal diagnosis of hereditary disorders,
besides identifying the carriers of genetic diseases.
By knowing the genetic basis of the disease, the individuals
can be advised on how to limit the transmission of the
diseases to their offsprings.
Also possible to treat genetic diseases by appropriate gene
therapies
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Some of the important genetic diseases for whichDNA analysis is used
• SINGLE-NUCLEOTIDE POLYMORPHISM
CYSTIC FIBROSIS
• Common fetal hereditary disease
• Produce thick and sticky mucus that clogs lungs and RT.
• Defect in CFTR gene that encodes cystic fibrosis
transmembrane regulator protein located on chromosome 7.
• DNA probe has been developed to identify this gene.
• Disease developed when 2 recessive genes are present.
• Fetal cells obtain from samples of amniotic fluid.
• Test can be done months before, it is possible to know whether
the offspring will be victim of CF. 27SSCP, Tumkur
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Sickle cell anaemia
• Characterized by the irregular, sickle shape of theerythrocytes.
• Results in to anemia damage to major organs.
• Occur due to single amino acid change in the β-chain ofhemoglobin.
• Glutamate at the 6th position of β-chain is replaced by
Valine.
• This single base mutation can be detected using restriction
enzyme MstII to cut DNA fragment (RFLP technique).
• Electrophoresis of formed DNA fragments.28SSCP, Tumkur
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Sickle cell anaemia
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TRIPLE REPEAT DISEASES• HUNTINGTON’S DISEASE
characterized by progressive deterioration of the nervous
system, particularly the destruction of brain cells.
older name was Huntington’s chorea; chorea means to dance
The gene responsible for this disease lies on chromosome
number 4, and is characterized by excessive repetition of the base
triplet CAG.
The triplet CAG encodes for the amino acid glutamine. It is
believed that the abnormal protein (with very high content of
glutamine) causes the death of cells in the basal ganglia.
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Fragile X syndrome
•
Due to a genetic defect in X chromosome (a sex chromosome)• Affects both males & females.
• Victims are characterized by mental retardation.
•Have three nucleotide bases (CGG) repeated again & again.
• These trinucleotide repeats block the transcription process
resulting in a protein deficiency.
•
This protein is involved in the normal function of the nervecells, & its deficiency results in mental retardation.
• A DNA probe has been developed for the detection of
fragile X syndrome in the laboratory.31SSCP, Tumkur
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Continued ……… • P53 GENE
• The gene p53 encodes for a protein with a molecular weight
53 kilodaltons.
• Thus, p53 is a cancer-suppressor gene and acts as a guardian
of cellular DNA.
• GENES OF BREAST CANCER
BRCAI and BRCAII function in a manner comparable to gene
p53 protein.
E.g., Gene for melanoma susceptibility, in humans are located
on chromosomes 1 and 9. 32SSCP, Tumkur
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DIABETES:-
• Clinical condition characterized by increased blood glucose
levels due to insufficient or inefficient insulin
• Type II diabetes is Maturity Onset Diabetes of The Young
(MODY ) found to have a genetic basis• A gene, synthesizing the enzyme glucokinase, located on
chromosome 7, is found to be defective in MODY patients.
•Single base pair mutation in the gene lead to the defective
glucokinase production.
• The glucokinase gene from normal and type II pts were
cloned and scanned with DNA probes.33SSCP, Tumkur
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OBESITY:-
•
It was in 1994, a group of workers identified a mutated genethat caused obesity in mice. Later, a similar gene was found in
humans also.
• The gene designated ob (for obese) is located on chromosome 6
in mouse. The DNA of ob gene contains 650 kb and encodes a
protein with 167 amino acids in adipose tissue. This protein is
responsible to keep the weight of the animals under control.
• Beside the ob gene, a few other genes like fat gene, tub gene
that might be associated with obesity have also been
discovered.
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Diagnostic Centers in India
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Continued ………
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Age of MotherFrequency of
Down Syndrome
Frequency of Any
Chromosomal Disorder
20
25
30
35
36
37
38
39
40
41
42
43
44
45
1 in 1667
1 in 1250
1 in 952
1 in 378
1 in 289
1 in 224
1 in 173
1 in 136
1 in 106
1 in 82
1 in 63
1 in 49
1 in 38
1 in 30
1 in 526
1 in 476
1 in 385
1 in 192
1 in 156
1 in 127
1 in 102
1 in 83
1 in 66
1 in 53
1 in 42
1 in 33
1 in 26
1 in 21
The American College of
Obstetrics and Gynecology
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REFERENCES Text book of Biotechnology by U. Satyanarayana. Pg: 173-184.
Pharmaceutical Biotechnology by Daan J A Crommelin andRobert D Sindelar. Pg:41-51.
Gene Biotechnology by S N Jogdand. Pg:83-89.
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/assist.shtml
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetest.shtml
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