Abstract

The aim of this study was to amplify the ancient DNA from a collection of 63
Romano-British neonatal skeletons, in order to determine their sex. The DNA testing
strategy has been designed to overcome the issues associated with ancient DNA
investigations, including degraded template DNA, inhibition of PCR, and modern
contaminants.
Ancient DNA has been extracted using a silica based method using the GENECLEAN
Kit for Ancient DNA. Three different PCR-Cleanup kits were investigated to
determine 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 Amelogenin
gene. The Amel-A and Amel-C primers used by Arnay-de-la-Rosa (2007) proved to be
ineffective, with multiple problems with non-specific binding and lack of
amplification of the target region. However, the amelogenin primers from the
AmpFISTR Identifiler kit proved more effective and were chosen for use in testing the
ancient material.
Modern DNA is used through the study to compare to the ancient material, in order
to allow for pre-testing of the techniques and optimisation of the PCR without the
loss of precious material. Modern DNA was also artificially degraded in order to
determine whether the PCR technique can amplify fractured DNA. In this
experimentation, the Identifiler primer PCR successfully amplified modern DNA that
had been degraded for 15 minutes in an ultrasonic bath, to a length of
approximately 100-500bp in size.
Measurements of the skeletons have also been taken in order to determine the
exact age of the neonates at time of death, to identify potential patterns in the
deaths of the individuals, and to identify a potential link between the age of the
individual at time of death and the probability of survival of the DNA within their
remains. Of the remains, 72% of the individuals were of an age of at least 38 prenatal
weeks, indicating a full term child considered as an infant death. 8% of individuals
provided an age range above 24 weeks, and were potentially stillbirths rather than
infant deaths, while 20% of the remains showed sufficient damage to prevent
complete age ranges that utilise both the length and width of the skeletal material
from being calculated and therefore may have been miscarriages of younger
foetuses or the fragmented remains of older infant remains.
Following the DNA analysis of the remains, three of the individuals provided
amplicons of the target region and therefore their sex could be determined. All three
of these individuals provided an XY male genotype. All other remains tested gave
negative results. Many of the results showed severe problems with PCR inhibition
even after the use of the PowerClean PCR Cleanup kit. Therefore, the conclusions
that can be drawn are limited. The potential for contamination to be the cause of
these three results is discussed, but the low sample size of positive results makes it
difficult to determine the source of the amplified DNA. Further work is suggested,
including more rigorous inhibitor removal methods and qPCR to provide a larger
sample size of positive neonatal DNA results, allowing for the validity of the results
to be assessed more thoroughly.
Inhibition and DNA degradation have proved to be the largest challenges faced in
this research, which is consistent with other research that utilises ancient DNA.
Further analysis of soil samples from the excavation and of the remains themselves
is suggested, to determine the type and quantity of inhibitory substances present.
Biochemical assessment to determine the level of preservation of the microstructure
of the bone, and the use of cloning and sequencing to identify DNA damage and rule
out contaminants is suggested.