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GENE THERAPY, RFLP, DNA Fingerprinting.pptx
- 1. Gene Therapy, RFLP, DNA Fingerprinting, Mutations Dr. Abhinav Manish MBBS, MD Asst. Prof. Dept. of Biochemistry GBCM
- 2. Gene Therapy • Genetic Diseases – Caused due to mutations. – Which leads to defective formation of gene product. • Deficiency of product- Hemophilia (factor VIII deficiency) • Defective metabolism for eg:- Galactosemia, Phenylketonuria, Hemochromatosis. • Enzyme defect due to genetic abnormalities. – 1st approved gene therapy- GLYBERA.(2012 discontinued)
- 3. Cntd. • Genetic disorders are managed by:- – Replacement of a deficient product. (factor VIII) – Dietary restrictions of such product which accumulates and causes disease. – But if the cause is deficiency of enzyme then it is difficult to replace as the sources of enzyme are limited, and also it is difficult to target to the specific organ where it should be utilized.
- 4. Pre-requisite of Gene Therapy • Several needs to be satisfied:- – Prognosis of disease – Genetic disease is due to well characterized single gene defect. Gene is isolated & fully characterized with defined regulatory elements. – Target cell and delivery method. – Enough evidence that gene encorporated via gene therapy will function. And product formed will not harmful.
- 5. Types of gene therapy Gene replacement Gene correction Gene augmentation (only method used currently)
- 6. Vectors used in the gene therapy • Retroviral vectors (carry DNA insert up to 9kbp) • Adenoviral vectors (carry up to 7.5 kbp) • Plasmid liposome complex
- 7. Introduction of the vector In- vivo gene therapy ( CFTR gene is introduced as aerosol in Respiratory tract) Ex-vivo gene therapy In- utero gene therapy
- 8. Outcome of Gene Therapy • It is feasible. • It is safe but long term outcome need to be evaluated. • Not immunological in nature (causes inflammation). • Results are variable but encouraging.
- 9. Current status of Gene Therapy • Conceptualized in 1972, Firstly tried in 1990, >1700 clinical trials have been conducted. • The following diseases have notable advances:- – SCID- Adenosine Deaminase deficiency – Chronic granulomatous diseases. – Hemophilia – Cancers – Neurodegenerative diseases (Parkinson’s disease, Huntington’s disease) – Hepatitis, HIV, Influenza – Coronary Artery Disease & Heart Failure – Blindness- Retinitis Pigmentosa – Muscular dystrophy etc. Disease Gene transferred by SCID By Retrovirus, ADA enzyme in ch.13&20 in lymphocyte Duchenne Muscular Dystrophy (DMD) By Retrovirus, Dystrophin gene on X. CF CFTR gene ch. 7, by Adenovirus Familial hypercholesterolemia LDL receptor gene on ch19, by retrovirus. Hemophilia A & B gene for factor 8 & 9 in fibroblast, by retrovirus Cancer Activation of p53, by Liposome Leber,s Hereditary Optic Neuropathy Adenoviral vector to retina.
- 10. RFLP- Restriction Fragment Length Polymorphism • It is an enzymatic procedure, for separation and identification of desired fragments of DNA. • Using RE enzymes fragments of DNA is obtained and the desired fragment is detected by using restriction probes. • Used to differentiated two organism by analysis of patterns derived from cleavage of their DNA. (VNTR are 10-60bps oligonucleotides sequences also k/s minisatellites, repeated 20 to 100 times & 1-4 bps short tandem repeats k/s microsatellites).
- 11. Cntd. • RFLP is a technique invented in 1984 by scientist Alec Jeffrey during research into hereditary diseases. • Genetic polymorphism is defined as the inherited genetic differences among individuals in over 1% of normal population. • The RFLP technique exploits these differences in DNA sequences to recognize and study both intraspecies and interspecies variation.
- 12. Procedure of RFLP • Step I: – Restriction digest – Extraction of desired fragments of DNA using restriction endonuclease (RE). – The enzyme RE has specific restriction site on the DNA, so it cut DNA into fragments. Different size of fragments are generated along with the specific desired fragments • Step II: – Gel electrophoresis – The digested fragment are run in electrophoresis to separate the fragments on the basis of length or size or molecular weight. – Different size of fragments form different bands.
- 13. • Step III: Denaturation – The gel is placed in sodium hydroxide (NaOH) solution for Denaturation so that single stranded DNA are formed. • Step IV: Blotting – single stranded DNA obtained are transferred into charge membrane i.e. Nitrocellulose paper by the process called capillary blotting or electro-blotting. • Step V: Baking and blocking – The nitrocellulose paper transferred with DNA is fixed by autoclaving. – Then the membrane is blocked by using bovine serum albumin or casein to prevent binding of labeled probe nonspecifically to the charged membrane.
- 14. • Step VII: Hybridization and visualization – The labeled RFLP probe is hybridized with DNA on the nitrocellulose paper. – The RFLP probes are complimentary as well as labeled with radioactive isotopes so they form color band. – These color bands visualize by autoradiography.
- 15. Application of RFLP • Determine the status of genetic diseases such as Cystic Fibrosis in an individual. • To determine or confirm the source of a DNA sample such as in paternity tests or criminal investigations. • In genetic mapping to determine recombination rates that show the genetic distance between the loci. • To identify a carrier of a disease-causing mutation in a family.
- 16. DNA Fingerprinting (DNA Profiling) • Each DNA profile is unique • Sequence of nucleotides, repeated again and again but different for person to person. • When the number is not known, variable, or irrelevant. It is known as VNTR. • When b/w 10-60 = minisatellites • When b/w 1-4= microsatellites or STRs. • Introns contain block of repeated STRs. • These STRs leads to variation in individuals.
- 17. • DNA fingerprinting technique was devised in 1985 by Alec Jaffrey at university of Leicester in England, while working on the sequence with in myoglobin gene. DNA is extracted from the sample. – SDS (sodium Dodecyl Sulphate detergent is used) – DNA is in nucleus (folded, molded, packed with proteins) – Proteinase K Ethanol Precipitation Cut with the restriction endonuclease. Separation of DNA Fragments via electrophoresis DNA fingerprint is obtained on nylon sheet by blotting technique and compared.
- 18. Restriction Enzyme based DNA Fingerprinting PCR- based DNA Fingerprinting Amplified Fragment Length Polymorphism(RFLP) STR (Short Tandem Repeat) Methods
- 19. Application of DNA Fingerprinting • Used in criminal, Medical, Paternity problems. • Violent murder or natural calamity, when recognition become difficult • Rape victims • Inheritance cases • Immigration • In Food industry • Cancer detection • Pattern of DNA profile is compared with victim and of suspect.
- 20. Mutation • DNA damage, caused by any agent, which leads to alteration in DNA sequence, which if replicated and transmitted to future generation, becomes permanent and such inherited change in base sequence of genomic DNA is K/S Genetic Mutation.
- 22. THANK YOU