Extracting maternal plasma during pregnancy for the purposes of molecular analysis has become increasingly common in recent times. The identification of fetal DNA in the maternal bloodstream represents a promising new hope for the development of non-invasive, prenatal diagnostic techniques which still encounter many problems in their application (Lo et al. 1998). The use of circulating cell-free fetal DNA found in maternal plasma enables forensic examination to be performed in paternity cases (Rong et al. 2012; Sarasola et al. 2006). Detection by means of circulating cell-free fetal DNA is capable of determining the gender of the foetus after 8 weeks of pregnancy (Colmant et al. 2013).
In this study, the respective DNA concentrations of circulating cell-free fetal DNA and maternal blood were compared, with significant differences being identified. This percentage indicates that the quantity of DNA derived from circulating cell-free fetal is high. DNA derived from circulating cell-free fetal DNA can be used as a specimen for the purposes of DNA examination by means of PCR method. This is due to the requirement that the volume of DNA specimens used in forensic DNA profiling must be at least 20 ng/ml (Notosoehardjo 1999).
The average purity of both maternal blood DNA or circulating cell-free fetal DNA is 1.79, allowing DNA to be used in PCR amplification (Muladno 2002). DNA purity can be classified as high if it falls within the range of 1.8–2.0. This study shows DNA purity to fall outside that range possibly because protein contamination might have occurred during the isolation of the DNA sample. The purity level of forensic DNA samples does not pose a significant problem if it remains above 1. The purity of DNA ranging from 1 to 2 for forensic DNA examination can still be used for DNA typing using PCR (Notosoehardjo 1999; Muladno 2002).
PCR is a sensitive method of DNA amplification. In forensic science, particularly the field of DNA typing, this method is also applied due to its ability to amplify small quantities of DNA (Kashyap et al. 2004). However, the limited amount of target DNA in certain circumstances does not affect the success of PCR. This research used several STR loci, such as loci vWA, TH01, D132317, D18S51, and D21S11, in addition to amelogenin genes for gender examination. It can be concluded from this study that the detection of forensic DNA of a length less than 400 bp was successful. The possibility of DNA fingerprinting using cell-free fetal DNA specimens was also confirmed. Nevertheless, the cell-free fetal DNA contained in certain samples failed the DNA fingerprinting process for one sample of circulating cell-free fetal DNA during the examination of loci D18S51 and D21S11. This occurrence might have been due to the fact that circulating cell-free fetal DNA in maternal plasma is, in general, shorter than maternal DNA (Rong et al. 2012). Failures in identification in loci D18S51 and D21S11 could also have been caused by problems associated with the amplification process or the occurrence of “false negative” results in the DNA multiplication process. These were possibly caused by the quality of circulating cell-free fetal DNA from the foetus or of fetal origin in the maternal plasma. Hence, the research samples in loci D18S51 and D21S11 were not successfully identified.
This research provided promising results for the use of circulating cell-free fetal DNA as a specimen of paternity examination in loci vWA, TH01, D13S317, D18S51, D21S11 and gender examination using amelogenin genes with non-invasive techniques. The study conducted by Wegner et al. (2009) was not able to successfully amplify autosomal fetal alleles from maternal plasma. Amelogenin was the only locus that was reliably amplified by the use of AmpFLSTR. Amelogenin revealed only fetal gender, while the amplification of other autosomal loci was sporadic and insufficient for reliable paternity testing (Wagner et al. 2009). The study reported here confirmed the success of this research as being relatively high: approximately 100% for loci of vWA, TH01, D13S317, 100% for amelogenin genes, 90% for loci D18251 and D21S11, giving an average success rate of 96%. This rate is relatively high considering the circulating cell-free fetal DNA. The results of this study are in accordance with those of a study conducted by Jiang et al. (Jiang et al. 2016) which proved that maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, possibly providing an alternative for forensic application in the future. This method could also be a unique means of detecting DNA loci with an average length of 100 bp (Rong et al. 2012).
According to a previous study conducted by Rong et al. (Rong et al. 2012), the Loci of vWA, TH01, D13S317, D18251, D21S11 and gender examination using amelogenin feature relatively long-base pairs which facilitate targeted locus examination. Locus D13S317 has a length of 169–201 bp, D18S51 is 290–266 bp in length, D21S11 is 203–259 bp in length, VWA is 127–167 bp in length, TH01 is 179–203 in length, while amelogenin genes are 106–112 bp in length. Long-base pair loci can be employed as paternity examination loci using cell-free fetal DNA as the examination material, although fetal cfDNA has reportedly also played this role for at least a decade (Ryan et al. 2013).
Paternity tests can be carried out using circulating cell-free fetal DNA (Ryan et al. 2013). There are three main reasons why cell-free DNA can be used for the purposes of paternity tests. The first is that circulating cell-free fetal DNA is derived from a foetus circulating in maternal blood even though at low concentration levels. Cell-free fetal DNA is essentially the DNA molecules outside the maternal DNA derived from the foetus whose use in DNA paternity tests has been sanctioned. Circulating cell-free fetal DNA inherits half of the maternal genetic sequences, thereby enabling it to be distinguished from the mother’s DNA (Wright 2009). This study could prove that there is allele conformity at loci D13S317, D18S51, D21S11, VWA, TH01 between fetal alleles and those derived from a mother and father in accordance with Mendel’s hereditary law.
Regarding the amount of human DNA, there are several different opinions about the minimum amount required for DNA analysis in forensic science. The minimum required amount for DNA typing in forensics ranges from 0.1 to 50 ng (Mandrekar et al. 2001). Another study showed that the DNA templates recommended for use in DNA typing contain 100–1000 ng (Kline et al. 2003). Meanwhile, a further piece of research confirmed that the minimum amounts of DNA used in forensic science are 50 ng and 20 ng respectively (Notosoehardjo 1999). STR method-based DNA testing could produce reliable results at minimum DNA concentrations of 0.5–2.5 ng (Kline et al. 2003). Although there are different opinions about the minimum amount of DNA that can be used in DNA analysis, in principle, the required amount in DNA forensic analysis depends on the necessity for and type of examination conducted. The Restriction Fragment Length Polymorphism (RFLP)-based forensic DNA examination requires relatively large amounts of about 100–1000 ng (Kline et al. 2003). The large amount of DNA used for RFLP tests is far from fresh, but the large amount of DNA is aimed to increase the likelihood of success in the management of DNA profiling. The STR test only requires a minimum DNA concentration ranging from 0.25–2 ng (Andelinović et al. 2005).
In addition, the amount or concentration of DNA for PCR-based DNA examination needs to be adequate, while its degraded condition should be at a minimum. Seriously degraded DNA has the potential to prevent primer attachment or aneling on the target DNA. Therefore, the quality of DNA represents a fundamental factor in the successful use of the PCR method (Yamada et al. 2002). The sensitivity of PCR is dependent on the function of the DNA’s cycle, amount and integrity. Low DNA levels in forensic investigations may sample up to a certain level that will not significantly affect the success of DNA profiling in forensic science, especially in STR tests. This is due to the amount of DNA required for the successful conducting of STR tests tending to be the lowest compared to other DNA-based tests. Consequently, the risk of failure of DNA amplification with this technique is relatively small (Chung et al. 2004).
