DNA Based Diagnosis of Genetic Diseases


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Diagnosis of genetic diseases


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Presented by Sharanabasappa I st 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 2 SSCP, Tumkur


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. 3 SSCP, Tumkur

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Continued …… 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. Inherited genetic defect can be diagnosed by identifying the alterations in the gene. 4 SSCP, Tumkur


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 5 SSCP, Tumkur


METHODS OF DNA ASSAY Nucleic acid hybridization a) radioactive detection system b) non – radioactive detection system DNA probes a) PCR in the use of DNA probes b) DNA probes & signal amplification DNA chip – microarray of gene probe 6 SSCP, Tumkur

Nucleic acid hybridization:

Nucleic acid hybridization Hybridization is the process of establishing a non-covalent , 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 7 SSCP, Tumkur

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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 identified 8 SSCP, Tumkur

Radioactive detection system :

Radioactive detection system 9 SSCP, Tumkur

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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 10 SSCP, Tumkur

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Continued …… Disadvantages of Radioactive detection system isotopes have short half life’s risks in handling requiring special laboratory equipments . 11 SSCP, Tumkur

Non-Radioactive detecting system:

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 12 SSCP, Tumkur

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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. 14 SSCP, Tumkur

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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 two separated single DNA strands can reassemble to form the original ds DNA. 16 SSCP, Tumkur

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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: Isolation of selected regions of genes Cloning of intact genes Producing from mRNA 17 SSCP, Tumkur

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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. 18 SSCP, Tumkur

PCR in the use of DNA probes:

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. 19 SSCP, Tumkur

DNA probes & signal amplification:

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. 20 SSCP, Tumkur

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Continued …… Separate the DNA target – DNA probe complex from the rest of the DNA molecules, & then amplify it. 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. 21 SSCP, Tumkur

DNA Chip – Microarray of Gene Probes :

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 22 SSCP, Tumkur

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Continued …… 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 23 SSCP, Tumkur

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Continued …… Applications Presence of mutations in a DNA sequence is identified. Genechip probe array has been successfully used for the detection of mutations in the p 53 & BRCA 1 genes (involved in cancer). Scientists are trying to develop Genechips for the entire genome of an organism. 24 SSCP, Tumkur

Importance of DNA in the Diagnosis of Genetic Diseases:

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. 25 SSCP, Tumkur

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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 26 SSCP, Tumkur

Some of the important genetic diseases for which DNA analysis is used:

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 the offspring will be victim of CF. 27 SSCP, Tumkur

Sickle cell anaemia :

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 β-chain of hemoglobin. 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. 28 SSCP, Tumkur

Sickle cell anaemia :

Sickle cell anaemia 29 SSCP, Tumkur


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. 30 SSCP, Tumkur

Fragile X syndrome:

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. 31 SSCP, Tumkur

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Continued ……… P 53 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. 32 SSCP, 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. 33 SSCP, Tumkur


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. 34 SSCP, Tumkur

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Diagnostic Centers in India 35 SSCP, Tumkur

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Age of Mother Frequency 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 37 SSCP, Tumkur

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Questions 38 SSCP, Tumkur


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/medicine/assist.shtml http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetest.shtml 39 SSCP, Tumkur

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