Presented by Sharanabasappa IIst M.Pharm. Dept.

of Pharmacology

Subject In Charge Dr. THIPPESWAMY B. S. M.Pharm., Ph.D Professor & Head of the Department DEPT. OF PHARMACOLOGY SSCP, Tumkur 1 SSCP, 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
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INTRODUCTION
Diagnosis of diseases due to pathogens or due to inherent genetic defects is necessary for appropriate treatment. Traditional diagnostic methods for parasite infections include microscopic examination, in vitro culture, & detection of antibodies in serum. And for genetic diseases, the procedures such as estimation of metabolites (blood/urine) & enzyme assays are used. These laboratory techniques are indirect & not always specific.
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DNA, being the genetic material of the living organisms, contains the information which contributes to various characteristic features of the specific organism.

Thus, the presence of a disease-causing pathogen can

be detected by identifying a gene or a set of genes of the organism. identifying the alterations in the gene.
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Inherited genetic defect can be diagnosed by

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: Alzheimers disease, arthritis, diabetes, cancer, and obesity
5) Mitochondrial
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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 noncovalent, sequence-specific interaction between two or more complementary strands of nucleic acids into a single hybrid.

There are two types of DNA hybridization techniques: a) Radioactive detection system b) Non-radioactive detection system

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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 identified
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Radioactive detection system

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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 Non hybridized probe DNA is washed away Presence of radioactivity in the hybridized DNA, detected by autoradiography.

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Disadvantages of Radioactive detection system
isotopes have short half lifes 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 than

chromogenic detection system

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DNA PROBE/GENE PROBE

Synthetic single stranded DNA molecule that can recognize and 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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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 two

separated single DNA strands can reassemble to form the

original ds DNA.
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Methods used to obtain DNA probes
Majority of DNA probes are chemically synthesized in the laboratory. 1) 2) 3) Many other ways are: Isolation of selected regions of genes Cloning of intact genes Producing from mRNA

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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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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 scanned simultaneously.

Advantages: Very rapid

Sensitive & Specific Simultaneous analysis of many DNA is possible
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Technique
Unknown 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 SSCP, Tumkur 23

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Applications
Presence of mutations in a DNA sequence is identified. Genechip probe array has been successfully used for the detection 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 Diagnosis of 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 advance

whether the individuals or their offsprings are at risk for

any genetic diseases.
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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 which DNA 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
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Sickle cell anaemia

Characterized by the irregular, sickle shape of the erythrocytes. Results in to anemia damage to major organs. Occur due to single amino acid change in the -chain of hemoglobin. Glutamate at the 6th position of -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.
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Sickle cell anaemia

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TRIPLE REPEAT DISEASES

HUNTINGTONS DISEASE characterized by progressive deterioration of the nervous older name was Huntingtons chorea; chorea means to dance The gene responsible for this disease lies on chromosome system, particularly the destruction of brain cells.

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 nerve

cells, & its deficiency results in mental retardation.

A DNA probe has been developed for the detection of fragile X syndrome in the laboratory.
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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. SSCP, 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.
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OBESITY: It was in 1994, a group of workers identified a mutated gene that 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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The American College of Obstetrics and Gynecology

Age of Mother
20 25 30 35 36 37 38 39 40 41 42 43 44 45 Frequency of Down Syndrome 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 Frequency of Any Chromosomal Disorder 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

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REFERENCES
Text book of Biotechnology by U. Satyanarayana. Pg: 173-184.

Pharmaceutical Biotechnology by Daan J A Crommelin and Robert D Sindelar. Pg:41-51.

Gene Biotechnology by S N Jogdand. Pg:83-89. http://www.ornl.gov/sci/techresources/Human_Genome/medici ne/assist.shtml

http://www.ornl.gov/sci/techresources/Human_Genome/medici ne/genetest.shtml

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