An addition to the disaster victim identification investigators toolkit?

by E.A.M. Graham, E.E. Turk and G.N. Rutty Forensic Science International : Genetics vol.2 (2008) pp.29-34

Mass fatality incidents

Disasters

• Environmental

• Medical

• Industrial

• Vehicle accident

• Terrorist event

• other

Requirement for the individualidentification of victims

Disaster victim identification(DVI) team

Example environmental event

Indian Ocean Tsunami : on 26th December 2004

-Death of over 200,000-Little or no fragmention but decomposition and putrefaction-Occurred due to the tropical climate

Primary identification criteria (in case of DVI)

• Odontology

• Fingerprinting

• DNA

• Observation of unique characteristics

- surgical prostheses

DNA profiling

• Can prove useful in allowing identification

• Re-association of fragmented, burnt or

decomposed corpses

• But successful of these is dependent upon

the collection and preservation

DNA profiling is the process of separating an individual's unique

DNA profiling

• Restriction fragment length polymorphism

(or RFLP)

• Allele-specific testing

- PCR : Polymerase Chain Reaction

- STRs : Short Tandem Repeats

two processes of DNA profiling in common use :

ref: http://www.kathyreichs.com/dnaprofiling.htm

-requires a relatively large sample of DNA -the fresher the better so this can be a drawback in criminal cases, where DNA is often taken from tissues that are degraded or contaminated by exposure to the elements

PCR: Polymerase Chain Reaction

ref: http://www.kathyreichs.com/dnaprofiling.htm

-can be done on a much smaller -less pure sample and less time than RFLP

but results in a much less precise profile

ref: http://www.kathyreichs.com/dnaprofiling.htm

- a PCR based allele-specific test - faster and more specific

Why preserve samples at room temperature?

• Usually, samples stored at -20 oc to halt the degradation processes but additional processing of samples is required

• Sometime refrigeration is not immediately available

• Transportation of samples from one country to another may required

Experiment

• Sample collection - obtained from patients donation of their two lower limbs (were

amputated due to chronic lower leg ischemia caused by diabetes)

[samples is muscle (soft tissue type)]

• Preservation methods - lysis storage

- transportation (LST) buffer

- Oragene TM DNA self-collection kit

• Experimental design

Experimental design

Pieces of muscle

Sample weighing1000, 500, 250, 100, 50, 25, 10 and 5 mgwere placed into Oragene TM collection pots

Sample weighing1000, 500, 250, 100 and 50 mgwere placed into 5 mL LST buffer

Sample weighing100, 50, 25, 10 and 5 mgwere placed into 1 mL LST buffer

DNA extraction was performed on each sample after 1, 2, 4, 12, 36 and 52 weeks

Qiagen DNA mini kit

DNA extraction

Incubation at 50 oc for 3 hour

1/25th Oragene TM purifier solution

Oragene TM purification protocol

Incubation on ice for 10 min centrifugation wash

Recover DNA (pellet form)

Re-hydration

Buffer of water100µL aliquots

Qiagen DNA mini kitUse blood/body fluids protocol(manufacturer’s instruction)

100µL aliquots

elution in 100 µL buffer AE

DNA quantification

1 µL of each extracted sample (duplicate)

Quantifiler Human DNA Quantification kit in a total reaction volume = 12.5 µL

Thermal cycling

7500 Real-Time PCR System (manufacturer’s instructions)

DNA profiling

DNA from all extracted samples

AmpF/STR ® SGM Plus ® PCR Amplification kit in a total reaction volume = 12.5 µL

1 ng template DNA

28 amplification samples Partial or failed DNA profile

re-amplification

Analysed by GeneMapper ID software

34 PCR cycles

PCR products of week1, 2, 4, 12 and 36 extracts

Separated and Visualised on an ABI PRISM ®377 DNA Sequencer

Fragment sizing by GeneScan® software

Allele designation by Genotyper® software

Separated and Visualised on an Applied Biosystems 3130 Genetic Analyser

PCR products of week 52 extracts

Results

Average result of real-time PCR quantification performed in duplicate for all samples extracted during a 52-week period

(ng/µL) (ng/µL)

Fig.1.Quantity of DNA recovered from muscle tissue stored in Oragene TM solution. Each bar represents the average quantity of DNA recovered from a 100 µL aliquot over the six time periods sampled. The error bars indicate the 95% confidence interval for each data set. (These data have been normalised by dividing the total DNA yield (ng) by the amount of tissue (mg) in each samplein order to compare the efficiency of each extraction.

Fig.2.Quantity of DNA recovered fromMuscle tissue stored in 5 mL LST buffer.

Fig.3.Quantity of DNA recovered from muscle tissue stored in 1 mL LST buffer.

Conclusion

• Quality of DNA recovered from muscle tissue stored in both Oragene TM and LST preservative buffers may begin to diminish after 6 months at room temperature.

• Quality of DNA recovered from tissue stored in LST buffer is not significantly reduced compared with that stored in Oragene TM collection pots.

• The yield of DNA per mg of tissue stored in 1 mL was greater than to 5 mL LST buffer.

• LST buffer is better suited to the preservation of small amounts (<100 mg) of tissue.

• Both buffer solutions have shown sufficient DNA preservation over a 12 month period of storage at room temperature.

Conclusion (cont.)• Oragene TM collection pots is superior to LST buffer in

recovery of high DNA yield, especially when stored in 5 mL LST buffer.

• DNA preservation in hard tissue (bone or teeth) is superior to soft tissues (muscle) especially when putrefaction but processing of hard tissue is extremely time-consuming and labour-intensive.

• Low-cost LST buffer and storage without refrigeration. (no additional processing of tissue is required)

• This system could also allow for automation of DNA extraction process by use of robotic platforms such as the Qiagen Biorobot, if required. (increase in sample throughput)

• This system allows for the collection of small pieces of muscle (or other soft tissue).

Conclusion (cont.)

• This system is fully portable and is compatible with bar-coding management systems.

• From results of both this work and previously work using similar preservation buffers shows that it is applicable to burnt remains and changes of decomposition.

• Promoted for DVI field work and is especially applicable in an incident involving disrupted body parts where traditional DNA samples or teeth and bone may not be readily available for identification and fragment re-association.