DNA fingerprinting & DNA footprinting
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Transcript of DNA fingerprinting & DNA footprinting
Submitted to Submitted by Prof. (Smt.)U.K.Chauhan SANJAY VERMA Deptt. Of Biotechnology M.Sc. Biotech IIIrd Sem.
A.P.S.U. Rewa (M.P.)
SEMINAR REPORT ON
DNA FINGERPRINTING &
DNA FOOT PRINTING
DNA FINGERPRINTING & DNA FOOT PRINTING 1. Introduction 2. History 3. Satellite DNA and Types of satellite DNA. 4. Probes used for DNA fingerprinting 5. RFLP (restriction fragment length polymorphism) 6. DNA fingerprinting in paternity case 7. Applications of DNA fingerprinting 8. DNA foot printing or EMSA 9. Principle of EMSA and variation of DNA foot printing A. Nuclease Protection foot printing B . Modification Protection foot printing 10. Applications of DNA foot printing 11. Conclusion
DNA fingerprinting (DNA profiling, DNA testing or DNA typing) is a forensic technique used to identify individuals by characteristic of their DNA. DNA fingerprinting uses repeated sequences that are highly variable number of tandem repeats. DNA fingerprinting is based on RFLP.
DNA foot printing also referred as gel shift assay, band shift assay or gel retardation assay is a common affinity electrophoresis technique used to study protein-DNA and protein-RNA interaction.
2. HISTORY DNA fingerprinting first developed and used by professor of genetic Alec Jeffreys at Leicester University U.K. in 1985.
The term of DNA fingerprinting was used by Jeffreys for MLP analysis. He had used the VNTR having GGGCAGGANG repeated sequence.
3. Satellite DNA
Satellite DNAs consist of short sequences about five to a few hundred base pairs in up to several million base pairs of DNA.
Types of satellite DNA. A.Minisatellite DNA - Minisatellite DNA or VNTR
(D1S7) sequence range from about 10 to 100 base pairs and found in sizeable cluster containing as many as 3000 repeats.
B.Microsatellite DNA- Microsatellite DNA or STR are the shortest sequence (1 to 5 base pair long) and are typically present in small clusters of about 10 to 40 base pair in length
4. Probes used for DNA fingerprinting
A. Single locus probe - A probe that is specific for the STR or VNTR sequence at one locus in the genome as is the case here is called a monolocus or single locus probe.
B. Multilocus probe - Multilocus probes are short length of
DNA, which bind to target DNA at several places and thus produce many bands - a pattern like ladder.
Single Locus Probe
5. RFLP (restriction fragment length polymorphism)
RFLP is a codominant DNA marker. DNA fingerprinting is
based on RFLP technique. A single base change within a restrictions site is a readily detectable genetic marker because the mutated site is no longer cleaved by the restriction enzyme. RFLPs have only two alleles: the site is present or absent.
RFLP (restriction fragment length polymorphism)
6. DNA fingerprinting in paternity case The DNA typing proceeds as follows: A . DNA samples are obtained from all three individual involved in paternity case.
B. The DNA is cut with restriction enzyme for the marker to be analyzed. C. Gel Electrophoresis. D. Southern Blotting. E. Hybridization by probe with a labeled monolocus STR or VNTR probe.
F. Autoradiography detection is then analyzed to compare the samples. G. Data interpretation
DNA fingerprinting in paternity case
7. Applications of DNA fingerprinting
A. Forensic analysis in murder, rape and other violent crimes.B. Population genetics studies.C. Proving pedigree status in certain breeds of horses for breed
registration purpose.D. Conservation biology studies .E. To confirm cell line identify in a cell line collection.F. For the detection of genetically modified organisms (GMOs).G. PCR using strain- specific primer to test for the presence of
pathogenic E. coli strain in food sources.
8. DNA foot printing or EMSA (electrophoretic mobility shift assay)
The gel retardation assay tests the ability of a protein to bind a radiolabel
DNA fragment as it migrate through a non-denaturing gel under the influence of an electric current. binding of protein will reduce the mobility of the DNA.
Variations of DNA foot printing -
A. Nuclease protection foot printing B. Modification protection foot printing
9. Principle of EMSA A mobility assay is electrophoretic separation of a protein-
DNA & protein-RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15 to 20 cm gel). The speed at which different molecules (and combination thereof) move through the gel is determined by their size and charge. The control lane (DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment.
A. Nuclease protection foot printing
The DNaseI foot printing assay measures the ability of a protein to protect a radiolabel DNA fragment against digestion by DNaseI. If a protein X binds the DNA at a particular site, then this protects that site against digestion by DNaseI, as a consequence, a gap or footprint appears in the ladder of DNA molecule.
Nuclease protection foot printing
B. Modification protection foot printing
DNA fragments are treated with limited amounts of dimethyl sulfate (DMS) so that a single G base is metylated in each fragment. If a protein X binds the DNA at particular site, then this protects guanines at that site from the action from dimethyl sulfate. Guanines that are protected by the bound protein X cannot be modified.
After removal of the protein, the DNA is treated with piperidine, which cuts at the modified nucleotide positions. Piperidine only cuts the strand that is modified.
Modification protection foot printing
10. Applications of DNA foot printing To analyze sequence-specific recognition of nucleic acid by
proteins and the management of cell growth and behavior. In study of regulation of gene expression.For studies on transcriptional regulation in bacteria.In the study of AU-rich element (ARE) and a member of
human Hu family RNA-binding protein (HuR) interaction. EMSAs using near-infrared fluorescence technology are used
to study of RNA processing, DNA replication, DNA repair mechanism, protein-DNA interaction and protein-RNA interaction.
11. Conclusion DNA fingerprinting is a potent weapon in forensic medicine
traditionally one of the of most accurate methods for placing an individual at scene of a crime has been a fingerprint. DNA foot printing provides a practical and efficient method in the quantitative study of RNA-protein interaction which plays an important role in many biological processes such as translational efficiency and mRNA stability.