Dna fingerprinting
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DNA Fingerprinting of plants . History,procedure of DNA fingerprinting, PCR and NON PCR technique like RAPD,SSR,RELPs, application of DNA fingerprinting, advantage and disadvantage of DNA fingerprinting.
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Dna fingerprinting
- 1. DNA FINGERPRINTING Presented By : Shikha Bhardwaj M.Pharma (Pharmacognosy)
- 2. DNA FINGERPRINTING • DNA fingerprinting was invented in 1984 by Professor Sir Alec jeffreys after he realised you could detect variations in human DNA in the form of these minisatellites. • It is a technique used especially for identification(as for forensic purposes) by extracting and identifying the base-pair pattern in an individual’s DNA. • Microsatellite :a set of short repeated DNA sequences at a particular locus on a chromosome, which vary in number in different individuals and so can be used for genetic fingerprinting.
- 3. How was the first DNA fingerprint produced?
- 4. Procedure of DNA fingerprinting in plants Isolation of DNA from plant: Plant part like leaves , roots, stem by removal of cell wall, nuclear membrane, around DNA and separation of DNA from cell debris, proteins, lipids and RNA by common CTAB method. Quality and quantity of isolated DNA was check is done by Uv-Visible spectroscopy. Denaturation of double strands at particular temperature . Annealing that includes the one primer binds with the 5’ end of one DNA strand and the other primer binds to 3’ end of its complementary strand.
- 5. • PCR based : RAPD(Random Amplification Polymorphic DNA) ISSR(inter simple sequence repeat) SSRs( Simple sequence repeats) • Non PCR based: RELP • PCR product were separated by electrophoresis in 1.5%(w/v) agarose gel using buffer. Different bands are produced. CHARACTERISTICS RAPD RELP AFLP PRINCIPLE DNA amplification restriction digestion DNA amplification DETECTION DNA staining Southern blotting DNA staining PRIMER REQUERMENTS Yes (random primer) none Yes(selective primer) PROBE REQUERMENT none Set of specific probes none
- 6. RAPD (Random Amplification Polymorphic DNA) • RAPD that is defined by differences between individuals in term of DNA regions either being or not being amplified in polymerase chain reaction primed by random oligonucleotide sequences. • It is a type of PCR reaction, but the segments of DNA that are amplified are random. • RAPD creates several arbitrary, short primers (8-12 nucleotide), then proceeds with the PCR using large template of genomic DNA. • Unlike traditional PCR analysis, RAPD does not require any specific knowledge of DNA sequence of target organism: the identical 10- primers will or will not amplify a segments of DNA, depending on positions that are complementary to the primer’s sequence.
- 7. RAPD involves following steps: 1. The DNA of a selected species is isolated. 2. An excess of selected decaoligonucleotide added. 3. Mixture is kept in PCR and subjected to repeated cycles of DNA denaturation-renaturation-DNAreplication. 4. Decaoligonuclotide will pair with the homologous sequence present at different locations in DNA. 5. DNA replication extend the decaoligonucleotide and copy the sequence continuous with the sequence with which the selected oligonucleotide has paired. 6. PCR cycles amplify the sequence of DNA. 7. Amplification of only those regions which are complementary to decaoligonucleotide.
- 8. 8.After amplification the DNA is subjected to gel electrophoresis. 9. The amplified DNA will form a distinct band. It is detected by ethidium bromide staining and visible fluorescence’s under U.V. light.
- 9. Advantage of RAPD • Efficiency and low expense. • It involves no radioactive assays. • Provide quick and efficient screening for DNA sequence based polymorphism at many loci.
- 11. Limitations of RAPD • Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segments is amplified from a locus that is heterozygous(1 copy) or homozygous(2copy). • Mismatch b/w the primer and the template may result in total absence in PCR product as well as in a merely decrease amount of the product. Thus RAPD effect difficult to interpret. • PCR is a enzymatic reactions, therefore the quality and concentration of template DNA, concentration of PCR component, and the PCR cycling conditions may greatly influence the outcomes.
- 12. Application of DNA fingerprinting • Individuality : like skin finger printing , DNA finger printing can help to distinguish one human being from another with exception of monozygotic twins. • Paternity/maternity Disputes : DNA fingerprinting can identify the real genetic mother, father and the offspring. • Human Lineage: DNA from various probables is being studied to find out human lineage. • Hereditary Diseases : the technique is being used to identify genes connected with hereditary diseases. • Forensics: detection of crime and legal pursuits. • Sociology: identify racial grips, their origin, historical migration and invasions.
- 13. Advantages and Disadvantages Advantage: DNA profiling is an ideal method for confirming an identity with absolute certainty. It’s easy and painless to obtain a specimen for testing. A thorough scientific test can be conducted in as little as 48hr DNA testing is affordable and reliable.
- 14. Disadvantage : A DNA test should be run multiple samples, at least twice. For diagnosis collect four samples and the lab runs every test twice to avoid false readings.
- 15. REFERENCE • www.yourarticlelibrary.com/dna/dna-fingerprinting- principles-and-techniques-of-dna-fingerprinting/12211 • www.yourgenome.com/what is a dna-fingerprint. • https://www.slideshare.net DNA Fingerprinting by Rachana Tiwari • E. Selvakumari, J.jenifer, Volume 5, 2017.Application of DNA Fingerprinting for Plant Identification. • Singh B.D. ‘Biotechnology: Expending Horizons’ Kalyani publishers ,2010. • Chawla H.S ‘Introduction To Plant Biotechnology’ Oxford and IBH publishing, third edition 2010.