Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

Abstract

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Fig 1. Schematic of the AMEL sense and antisense primer sets.

The primer sequences are described in Table 1. A and B show sense and antisense primer sets, respectively. Yellow and pink boxes represent allele-specific primers. Orange boxes serve as common sense or antisense counter primers. Gray boxes indicate sense and antisense DNA templates. Blue letters show the 3ʹ-terminal bases in allele-specific primers, whereas red letters show C↔G transversion polymorphisms in sense and antisense DNA templates. Dotted boxes indicate base pairings at SNP sites between allele-specific primers and sense or antisense DNA templates.

Fig 2. Assessment of the quality of mock forensic samples.

Black and white columns show 129/41 bp and 305/41 bp Q-ratios, respectively. The Q-ratios correlate with the amount of UV radiation. Both female and male mock forensic samples with no UV exposure produced Q-ratios close to 1. Mock forensic samples subjected to high levels of UV irradiation yielded 129/41 bp and 305/41 bp ratios in the ranges of 0.00–0.38 and 0.00–0.038, respectively. Mean values ± SD are from triplicate assays.

Fig 3. Sex determination based on a novel PCR-APLP method using 1 ng of template DNA.

Lanes 1–4 correspond to female DNA, lanes 5–8 to male DNA, and lane 9 to the negative control. A and B show results of sex determination using the sense and antisense primer sets, respectively. M indicates the 10-bp ladder marker. The results were reproduced in three independent assays.

Fig 4. Sensitivity measurement by PCR-APLP using a dilution series of female and male DNA.

Lanes 1–6 correspond to a serial dilution of female DNA, lanes 7–12 to a serial dilution of male DNA, and lane 13 to the negative control. A and B show results of sensitivity analysis using the sense and antisense primer sets, respectively. M indicates the 10-bp ladder marker. The results were reproduced in three independent assays.

Fig 5. Robustness evaluation of a PCR-APLP method using UV-irradiated template DNA.

A and B show the results of robustness analysis using the sense and antisense primer sets, respectively. Lanes 1–5 correspond to female DNA damaged by 0, 0.5, 1.0, 5.0, and 10 J UV irradiation; lanes 6–10 to male DNA damaged by 0, 0.5, 1.0, 5.0, and 10 J UV irradiation; and lane 11 to the negative control. Lanes 1 and 6 are female and male positive controls, respectively. M indicates the 10-bp ladder markers. The results were reproduced in three independent assays.

Fig 6. Sex determination by PowerPlex^® ESX17 Fast using UV-irradiated template DNA.

A and C show female and male DNA with no UV irradiation, respectively. B and D show UV irradiation-exposed female (0.5 J) and male (0.2 J) DNA, respectively. The results were reproduced in three independent assays.

Fig 7. Sex determination by the PowerPlex^® Fusion system using UV-irradiated template DNA.

A and C show female and male DNA with no UV irradiation, respectively. B and D show UV irradiation-exposed female (1.0 J) and male (0.5 J) DNA, respectively. The results were reproduced in three independent assays.

Fig 8. DNA sexing of Jomon samples by our method.

A and B show the results of sex determination using the sense and antisense primer sets, respectively. M indicates the 10-bp ladder marker. The results were reproduced in three independent assays. Typical results of bidirectional analysis are shown because amounts of template DNA were not constant among ancient samples.