MSC IV SEMESTER_ DNA Profiling -PCR.pdf

DNA Profiling -PCR

1. DNA Profiling

2. Kary Mullis

3. PCR through ages

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

8. https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-
molecular-biology/pcr-education/pcr-thermal-cyclers.html

9. https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-
molecular-biology/pcr-education/pcr-thermal-cyclers.html

10. https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-
molecular-biology/pcr-education/pcr-thermal-cyclers.html

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
Ca
2+
Spermineandspermidine(
polyamines)
Urea

24. SUBSTRATE
Textile dyes (Indigo dye
used in denim)
Fabrics
(Tannic Acid)
Environmental
Samples Soil
(Humiccompounds
Heavy metals)
Food
Constituents
(Organic compounds
Phenolic compounds
Glycogen
Fats
Ca
2+)

25. Other
Inhibitors
Chelexresin
Salts
Guanidine
Proteases
Organic solvents
Phosphate buffers
Detergents (such asSodium Dodecyl Sulfate(SDS))
Powder
(glove powder)
Laboratory
plastic ware
(PCR tubes)
Anticoagulants
(EDTA and
heparin)
Plant and food products
(Pollen
Cellulose
Plant polysaccharides
Ca
2+)

26. HISTORICAL PERSPECTIVE
VNTR

27. DNA Analysis
Flowchart
PCR based assays

28. forensic PCR tests was based on identification
ofhuman 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
highdiscriminating 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

36. AmpFℓSTR (Applied Biosystems)

40. This Photo by Unknown Author is licensed under CC BY-SA