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).

Identification of Human Remains by DNA Analysis of the

gastrointestinal contents of Fly Larvae

Journal of Forensic Sciences 2012 (wiley.com) Presented by

Narmeen ArshadFUUAST

19th March 2014

Out Line: Case Report Background Methodology Discussion References

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.

Forensic Genetic AnalysisPossible sources of DNA

• Blood• Semen• Saliva• Urine• Feces• Perspiration• Bone• Tissue

Forensic Entomology:

Forensic entomology is the application and study of insect and other arthropod biology to

criminal matters.

Postmortem Interval:

Time b/w the death & discovery of the corpse.

METHODOLOGY

Working with evidences :

• Fly Larvae – Maggots – Burnt Body.• Alleged Father.

Procedure • DNA Finger Printing• Amelogenin PCR• Data Interpretation

Maggots

DNA from Larvae Gut was extracted by Following Procedure :

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.

Southern blot1. Restriction enzyme digestion2. Agarose gel electrophoresis3. Transfer DNA onto membrane4. Hybridized with probe

RFLP

RestrictionEnzyme 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 bandsof different mobility

PCR

Multiplex kit --- AMPFistr Identifiler

Kit Contents:

•Allelic Ladders for Genotyping

•PCR Component Mix

•Primer Mix

•Positive Control DNA Sample

CAPILLARY ELECTROPHORESISABI PRISM® 310 Genetic Analyzer

Automated gel pouring

Automated sample injection

Capillary electrophoresis with multi-color detection capabilities

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler trayOutlet buffer

Injection electrode

Inlet buffer

D8S1179 D21S11 D7S820 CSF1PO

D3S1358TH01

D13S317 D16S539 D2S1338

D19S433 D18S51TPOX

VWA

AMEL D5S818 FGA

GS500 LIZ size standard

6FAM (blue)

VIC (green)

NED (yellow)

PET (red)

LIZ (orange)

AmpFlSTR® Identifiler™

STR Typing

•What ?

•Why ?

Repetitive sequence within intron

• Short repeated DNA sequence within intron• Present in different chromosomal locations• Unknown functions

Minisatellites or microsatellite

Exon Exon ExonIntron Intron

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

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

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.

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

STR Marker #Alleles Random match probability (FBI Caucasian)CSF1PO 11 0.112FGA 19 0.036TH01 7 0.081TPOX 7 0.195VWA 10 0.062D3S1358 10 0.075D5S818 10 0.158D7S820 11 0.065D8S1179 10 0.067D13S317 8 0.085D16S539 8 0.089D18S51 15 0.028D21S11 20 0.039

Product 0.000000000000001683

One in 594,059,679,247,5401 in 594 trillion

Probability of a Random Match Using 13 CODIS STR Markers

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

Findings :

• Amelogenin PCR ---- Female Corpse• Probability of paternity testing -----

99.685 %• Results were later verified ----- DNA

analysis of bone, possible after many attempts.

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.

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/

Reference Papers