Sex determination of Romano-British neonatal remains via advanced biomolecular and anthropological techniques
Creators: name: Jackson, Robert
Publisher: name: University of Lincoln
Year: 2023
Subjects: F400 - Forensic &amp; archaeological sciences

description: The aim of this study was to amplify the ancient DNA from a collection of 63Romano-British neonatal skeletons, in order to determine their sex. The DNA testingstrategy has been designed to overcome the issues associated with ancient DNAinvestigations, including degraded template DNA, inhibition of PCR, and moderncontaminants.Ancient DNA has been extracted using a silica based method using the GENECLEANKit for Ancient DNA. Three different PCR-Cleanup kits were investigated todetermine their effectiveness in the removal of PCR inhibitors. The PowerClean kit(MoBio Laboratories) proved the most effective, and was used throughout.Two different sets of primers were used to provide amplicons from the Amelogeningene. The Amel-A and Amel-C primers used by Arnay-de-la-Rosa (2007) proved to beineffective, with multiple problems with non-specific binding and lack ofamplification of the target region. However, the amelogenin primers from theAmpFISTR Identifiler kit proved more effective and were chosen for use in testing theancient material.Modern DNA is used through the study to compare to the ancient material, in orderto allow for pre-testing of the techniques and optimisation of the PCR without theloss of precious material. Modern DNA was also artificially degraded in order todetermine whether the PCR technique can amplify fractured DNA. In thisexperimentation, the Identifiler primer PCR successfully amplified modern DNA thathad been degraded for 15 minutes in an ultrasonic bath, to a length ofapproximately 100-500bp in size.Measurements of the skeletons have also been taken in order to determine theexact age of the neonates at time of death, to identify potential patterns in thedeaths of the individuals, and to identify a potential link between the age of theindividual at time of death and the probability of survival of the DNA within theirremains. Of the remains, 72% of the individuals were of an age of at least 38 prenatalweeks, indicating a full term child considered as an infant death. 8% of individualsprovided an age range above 24 weeks, and were potentially stillbirths rather thaninfant deaths, while 20% of the remains showed sufficient damage to preventcomplete age ranges that utilise both the length and width of the skeletal materialfrom being calculated and therefore may have been miscarriages of youngerfoetuses or the fragmented remains of older infant remains.Following the DNA analysis of the remains, three of the individuals providedamplicons of the target region and therefore their sex could be determined. All threeof these individuals provided an XY male genotype. All other remains tested gavenegative results. Many of the results showed severe problems with PCR inhibitioneven after the use of the PowerClean PCR Cleanup kit. Therefore, the conclusionsthat can be drawn are limited. The potential for contamination to be the cause ofthese three results is discussed, but the low sample size of positive results makes itdifficult to determine the source of the amplified DNA. Further work is suggested,including more rigorous inhibitor removal methods and qPCR to provide a largersample size of positive neonatal DNA results, allowing for the validity of the resultsto be assessed more thoroughly.Inhibition and DNA degradation have proved to be the largest challenges faced inthis research, which is consistent with other research that utilises ancient DNA.Further analysis of soil samples from the excavation and of the remains themselvesis suggested, to determine the type and quantity of inhibitory substances present.Biochemical assessment to determine the level of preservation of the microstructureof the bone, and the use of cloning and sequencing to identify DNA damage and ruleout contaminants is suggested.