DNA Diagnosis of Genetic Diseases

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



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



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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http://en.wikipedia.org/wiki/Non-covalent


http://en.wikipedia.org/wiki/Nucleic_acid

http://en.wikipedia.org/wiki/Hybrid_(biology)

http://en.wikipedia.org/wiki/Hybrid_(biology)

http://en.wikipedia.org/wiki/Nucleic_acid






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



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 &amplified. 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



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





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



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



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



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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http://www.acog.org/publications/patient_education/bp094.cfm



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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DNA Diagnosis of Genetic Diseases