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Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in
Kimiko Aoki1,2, Hiroyuki Tanaka1, Takashi Kawahara1
1Anti-doping Research Laboratory, Japan Chemical Analysis Center, 6-39-4, Minami Senju, Arakawa-ku, Tokyo, 116-0003, Japan.
Journal of Clinical Medicine Research
|June 16, 2018
Summary
This study developed a new mitochondrial DNA typing assay for urine, effective for verifying sample origins even with trace DNA amounts. Blood DNA is not suitable as a reference for urine sample verification.
Area of Science:
- Forensic Science
- Genetics
- Biochemistry
Background:
- Standard urine sample identification in doping control relies on nuclear DNA (nDNA) short tandem repeat (STR) analysis.
- Urine contains very low nDNA concentrations, often insufficient for STR analysis after cryopreservation.
- This study addresses the need for an assay capable of typing mitochondrial DNA (mtDNA) from trace amounts of DNA in urine, particularly for Japanese populations.
Purpose of the Study:
- To develop a multiplexed assay for urine mitochondrial DNA typing from limited DNA samples.
- To assess the assay's efficacy for personal identification and verification of urine samples.
- To evaluate the applicability of the assay for Japanese populations.
Main Methods:
- A multiplexed suspension-array assay was developed using oligo-tagged microspheres (Luminex MagPlex-TAG).
- The assay targets C-stretch length in hypervariable regions 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel.
- The assay was validated using cryopreserved urine samples (n=63) and fresh blood samples (n=150).
Main Results:
- The assay achieved a discriminatory power of 0.960 for the Japanese population, categorizing subjects into 62 types using blood DNA.
- The detection limit for cryopreserved urine was as low as 0.005 ng of nDNA.
- Discrepancies in C-stretch patterns between blood and urine samples were observed in 5 out of 63 pairs.
Conclusions:
- The developed assay provides valuable information for verifying urine sample origins using minimal DNA.
- Mitochondrial DNA typing from urine is feasible even with trace amounts of DNA.
- Blood DNA cannot be reliably used as a reference for urine sample verification with this assay.
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