Original scientific paper

Validation of Testing and Interpretation Protocols for Low Template DNA Samples Using AmpFSTR® Identifiler®

Theresa Caragine ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA
Rebecca Mikulasovich ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA
Jeannie Tamariz ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA
Ewelina Bajda ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA
James Sebestyen ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA
Howard Baum ; Office of Forensic Sciences, New Jersey State Police,, New Jersey, NJ, USA
Mechthild Prinz ; Office of Chief Medical Examiner of the City of New York, The Department of Forensic Biology, New York, NY, USA

Abstract

Aim To test the reliability, robustness, and reproducibility
of short tandem repeat (STR) profiling of low template
DNA (LT-DNA) when employing a defined set of testing
and interpretation parameters.
Methods DNA from known donors was measured with
a quantitative real time polymerase chain reaction (PCR)
assay that consistently detects less than 1 pg/μL of DNA
within a factor of 0.3. Extracts were amplified in triplicate
with AmpFSTR® Identifiler® reagents under enhanced PCR
conditions. Replicates were examined independently and
alleles confirmed using a consensus approach. Considering
observed stochastic effects inherent to LT-DNA samples,
interpretation protocols were developed and their accuracy
verified through examination of over 800 samples.
Results Amplification of 100 pg or less of DNA generated
reproducible results with anticipated stochastic effects.
Down to 25 pg of DNA, 92% or more of the expected alleles
were consistently detected while lower amounts
yielded concordant partial profiles. Although spurious alleles
were sometimes observed within sample replicates,
they did not repeat. To account for allelic dropout, interpretation
guidelines were made especially stringent for
determining homozygous alleles. Due to increased heterozygote
imbalance, stutter filters were set conservatively
and minor components of mixtures could not be resolved.
Applying the resultant interpretation protocols, 100% accurate
allelic assignments for over 107 non-probative casework
samples, and subsequently 319 forensic casework
samples, were generated.
Conclusion Using the protocols and interpretation guidelines
described here, LT-DNA testing is reliable and robust.
Implementation of this method, or one that is suitably verified,
in conjunction with an appropriate quality control
program ensures that LT-DNA testing is suitable for forensic
purposes.

Keywords

forensic science; Low Template DNA (LT-DNA); Short Tandem Repeat (STR); Polymerase Chain Reaction (PCR), Stochastic Effects; Interpretation; IdentifilerTM

Hrčak ID:

40673

URI

https://hrcak.srce.hr/40673

Publication date:

15.6.2009.

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