Identification of Human Remains by DNA Analysis of the gastrointestinal contents of Fly Larvae A case Report that has been explained in form of presentation.

2. O Rabb! Open my heart, ease my task, and
loosen the knot from my tongue, so that they
might fully understand my speech (Al TAHA).

4. Out Line:
Case Report
Background
Methodology
Discussion
References

5. Case Report :
•Badly burned body --- Corpse obtained.
•Face & neck colonized ----- Fly Larvae.
•Soft Tissues ------Not available.
•Feet & hands ----Missing.
•Gender discrimination ----Not possible.
•Small fraction of ----Burnt Liver.
•Burned Bones.
•Only Evidence ---Graduation Ring.
•Woman --- Reported Missing (10 days
earlier).
•Genetic Profile --- Fragment of liver---
Unsuccessful.
•Alleged Father.

6. Forensic Genetic Analysis
Possible sources of DNA
• Blood
• Semen
• Saliva
• Urine
• Feces
• Perspiration
• Bone
• Tissue

7. Forensic Entomology:
Forensic entomology is the application and
study of insect and other arthropod biology to
criminal matters.

8. Postmortem Interval:
Time b/w the death &
discovery of the corpse.

9. METHODOLOGY

10. Working with evidences :
• Fly Larvae – Maggots – Burnt Body.
• Alleged Father.
Procedure
• DNA Finger Printing
• Amelogenin PCR
• Data Interpretation

11. Maggots

13. DNA from Larvae Gut was extracted
by Following Procedure :

14. DNA from alleged Father
• Buccal Sample collected.
• DNA extracted by Chelex protocol.
Five µl Buccal Sample + 200 µl of 5% Chelex 100.
Highly-purified control DNA extracted with phenol-
chloroform.

15. Southern blot
1. Restriction enzyme digestion
2. Agarose gel electrophoresis
3. Transfer DNA onto membrane
4. Hybridized with probe
RFLP
Restriction
Enzyme site 1. PCR amplify using flanking
fluorescent primers
2. Capillary electrophoresis
RFLP-based
DNA fingerprinting
PCR-STR based
DNA fingerprinting
PCR product of different sizes
Fluorescent peaks of different mobility
Radioactive bands
of different mobility

16. PCR
Multiplex kit --- AMPFistr Identifiler
Kit Contents:
•Allelic Ladders for
Genotyping
•PCR Component
Mix
•Primer Mix
•Positive Control
DNA Sample

18. CAPILLARY ELECTROPHORESIS
ABI PRISM® 310 Genetic Analyzer
Automated gel
pouring
Automated sample
injection
Capillary electrophoresis with multi-color
detection capabilities

19. ABI Prism 310 Genetic Analyzer
capillary
Syringe with polymer
solution
Autosampler trayOutlet
buffer
Injection
electrode
Inlet
buffer

20. D8S1179 D21S11 D7S820 CSF1PO
D3S1358
TH01
D13S317 D16S539 D2S1338
D19S433 D18S51
TPOX
VWA
AMEL D5S818 FGA
GS500 LIZ size standard
6FAM
(blue)
VIC
(green)
NED
(yellow)
PET
(red)
LIZ
(orange)
AmpFlSTR®
Identifiler™

22. STR Typing
•What ?
•Why ?

23. Repetitive sequence within intron
• Short repeated DNA sequence within intron
• Present in different chromosomal locations
• Unknown functions
Minisatellites or microsatellite
Exon Exon ExonIntron Intron

24. Mini. versus microsatellite
• No. of repeats varies among individuals
– VNTR (Variable Number of Tandem Repeats)
• Minisatellites
• Microsatellites
• Minisatellites
– Repeat unit length – 6-100 bases
– Two to several hundred repeats at each minisatellite
• Microsatellites
– Repeat unit length: 1-7 bases
– 5 to 100 repeats at each microsatellite
– Thousands of different microsatellites, randomly scattered
throughout genome
http://www.wellcome.ac.uk/en/genome/genesandbody/hg07f006.html

25. Short Tandem Repeats (STR)
• Each individual’s repeats is polymorphic in length
– For example:
• 25 % of population have 56 repeats in an STR
• 50 % have 58 repeats
• 25 % have 60 repeats
– Repeats can be 2 – 7 bp
• Repeat length at different STRs used as fingerprint.
– Universal: can be used to amplify DNA from any individual

26. The CODIS System
• National DNA Databank: CODIS
• CODIS = Combined DNA Index System
• In October 1998 the FBI's National DNA Index
System (NDIS) became operational.
• 13 CODIS Core STR Loci.

27. CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
13 CODIS Core STR Loci
AMEL
AMEL
Sex-typing
Position of Forensic STR Markers on Human
Chromosomes
Penta E
Penta D
D2S1338
D19S433

28. STR Marker #Alleles Random match probability (FBI Caucasian)
CSF1PO 11 0.112
FGA 19 0.036
TH01 7 0.081
TPOX 7 0.195
VWA 10 0.062
D3S1358 10 0.075
D5S818 10 0.158
D7S820 11 0.065
D8S1179 10 0.067
D13S317 8 0.085
D16S539 8 0.089
D18S51 15 0.028
D21S11 20 0.039
Product 0.000000000000001683
One in 594,059,679,247,540
1 in 594 trillion
Probability of a Random Match Using 13
CODIS STR Markers

30. Why STRs are Preferred
Genetic Markers ?
• Rapid processing is attainable
• Abundant throughout the genome
• Highly variable within various populations
• Small size range allows multiplex development
• Discrete alleles allow digital record of data
• Allelic ladders simplify interpretation
• PCR allows use of small amounts of DNA material
• Small product size compatible with degraded DNA

32. Findings :
• Amelogenin PCR ---- Female Corpse
• Probability of paternity testing -----
99.685 %
• Results were later verified ----- DNA
analysis of bone, possible after many
attempts.

33. Discussion:
• Accuracy
• Reliability
• Generation of good quality DNA
• Storage of Maggots
• Each maggot was processed
separately, same results obtained
• First reported case on forensic entomology in
Mexico for victim identification.

34. References
• Molecular Searching Techniques http://tinyurl.com/bfbeal
• DNA Diagnostics Center http://www.forensicdnacenter.com/dna-str.html
• Brief History of Forensic DNA Typing http://tinyurl.com/dmbgvl
• Wikipedia (easy reading for general principles)
http://en.wikipedia.org/wiki/DNA_fingerprinting
• CODIS (FBI) http://www.fbi.gov/hq/lab/codis/index1.htm
• DNA fingerprinting protocols (very detailed) by Applied Biosystems and population
genetics http://docs.appliedbiosystems.com/pebiodocs/04323291.pdf
• Southern blot Flash http://www.pbs.org/wgbh/nova/sheppard/labwave.html
• PCR principle
http://www.wellcome.ac.uk/en/genome/technologies/hg17b009.html
• http://www.cstl.nist.gov/biotech/strbase/

35. Reference Papers