Recommended
More Related Content
Similar to MSC IV SEMESTER_ DNA Profiling -PCR.pdf
Similar to MSC IV SEMESTER_ DNA Profiling -PCR.pdf (20)
More from Suchita Rawat
More from Suchita Rawat (20)
Recently uploaded
Recently uploaded (20)
MSC IV SEMESTER_ DNA Profiling -PCR.pdf
- 2. Kary Mullis
- 4. ● Very small amounts of DNA template ● DNA degraded to fragments ( few hundred base pairs) can serve as effective templates for amplification. ● Large numbers of copies of specific DNA sequences can be amplified(multiplex PCR reactions) ● Contaminant DNA (from fungal and bacterial sources) will not amplify because human-specific primers. ● Commercial kits are now available for easy PCR reaction setup and amplification
- 5. ● The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA. ● Amplification may fail due to sequence changes in the primer-binding region of the genomic DNA template. ● Contamination from other human DNA sources besides the forensic evidence at hand or previously amplified DNA samples is possible without careful laboratory technique and validated protocols
- 11. Controls Used to Monitor PCR: Control negative/ control positive
- 12. Primer
- 13. PCR Primer Improvements ✓ use of fluorescent dye tags ✓ non-nucleotide linkers to alter the electrophoretic mobility of the PCR product ✓ extra forward or reverse primers to cover multiple primer binding site possibilities ✓ Extra (degenerate) primer ✓ high-stability analogs (Increases primer annealing stability)
- 14. DNA polymerase AmpliTaq Gold DNA Polymerase pre-incubation of 95°C, usually for 10 or 11 minutes
- 15. Other Taq Polymerase Bio-X-Act Short (Bioline, London, UK) ExTaq Hot Start (Takara Bio Inc., Shiga, Japan) ✓ KAPA2G Robust (KAPA Biosystems, Cape Town, South Africa) ✓ OmniTaq (DNA Polymerase Technologies, St. Louis, MO, USA) ✓ PicoMaxx High Fidelity (Stratagene, La Jolla, CA, USA) ✓ rTth (Applied Biosystems) ✓ Taq, and Tth (Roche Diagnostics, Mannheim, Germany
- 16. PCR
- 17. Hot start PCR
- 18. NESTED PCR
- 19. Multiplex PCR
- 20. RT PCR
- 21. Real time qPCR
- 22. PCR Inhibition : Internal, or those found in body fluids. Substrates, or those arising from the materials on which the blood stain or other source of DNA has been deposited. Other sources, such as reagents and materials used in the analysis.
- 23. INTERNAL CONTAMITANTS Heme and its derivatives Immunoglobulin G Bacteria/Microorganisms melanin Ca2+ Spermine and spermidine ( polyamines) Urea
- 24. SUBSTRATE Textile dyes (Indigo dye used in denim) Fabrics (Tannic Acid) Environmental Samples Soil (Humic compounds Heavy metals) Food Constituents (Organic compounds Phenolic compounds Glycogen Fats Ca2+)
- 25. Other Inhibitors Chelex resin Salts Guanidine Proteases Organic solvents Phosphate buffers Detergents (such as Sodium Dodecyl Sulfate (SDS)) Powder (glove powder) Laboratory plastic ware (PCR tubes) Anticoagulants (EDTA and heparin) Plant and food products (Pollen Cellulose Plant polysaccharides Ca2+)
- 27. DNA Analysis Flowchart PCR based assays
- 28. forensic PCR tests was based on identification of human leukocyte antigen (HLA). HLA loci, DQ-Alpha
- 29. AmpFLPs (amplified fragment poymorphisms), examples that were evaluated for use in crime laboratories included: •D1S80 •YNZ 22 (D17S5) •3'ApoB
- 30. Short Tandem Repeats (STR) The selection of specific loci • The loci have a high discriminating power. •The loci are stable in evidence samples. •There is a consensus panel of core loci for use in DNA databases.
- 33. Scientific Working Group for DNA Analysis Methods (SWGDAM) Y-STR loci: •DYS19 •DYS385 A/B •DYS389I •DYS389II •DYS390 •DYS391 •DYS392 •DYS393 •DYS438 •DYS439 •DYS19
- 34. Factors in deciding the loci included in multiplex PCR: •Locus size and overlap •Number of alleles •Thermal cycling parameters •Primers •Dyes
- 40. This Photo by Unknown Author is licensed under CC BY-SA