forensic sciences (dna fingerprinting) str typing - case report
DESCRIPTION
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.TRANSCRIPT
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