A real-time PCR-based amelogenin Y allele dropout assessment model in gender typing of degraded DNA samples
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Allelic dropout due to stochastic variation in degraded small quantity DNA appears to be one of the most serious genotyping errors. Most methods require PCR replication to address this problem. The small amounts of valuable samples are often a limitation for such replications. We report a real-time PCR-based amelogonin Y (AMELY) allele dropout estimation model in an AMEL-based gender typing. We examined 915 replicates of AMELY-positive modern male DNA with varying amounts of DNA and humic acid. A male-specific AMEL fragment (AMELy) dropped out in 143 genuine male replicates, leading to gender typing errors. By graphing a scatter plot of the crossing point versus the end cycle fluorescence of the male replicates, a standard graph model for the estimation of the AMELy allele dropout was constructed with the dropout-prone and dropout-free zones. This model was then applied to ancient DNA (aDNA) samples. Nine samples identified as female were found in the dropout-prone zone; with higher DNA concentrations, six were shifted to the dropout-free zone. Among them, two female identifications were converted to male. All the aDNA gender was confirmed by sex-determination region Y marker amplification. Our data suggest that this model could be a basic approach for securing AMELy allele dropout-safe data from the stochastic variation of degraded inhibitory DNA samples.
KeywordsAMELY allele dropout Ancient bone Real-time PCR Melting curve analysis Amelogenin
We thank Jehyeok Lee and Yeong-mi Chang for their kind assistance in administration work and purchase of laboratory materials. We also thank Jee-hyun Yoon for the maintenance of the real-time PCR system. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0021367).
All sampling was done according to Korean laws and ethical standards.
Conflict of interest
The authors declare that there is no conflict of interest.