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Determination of the inheritance of the paternal allele Amplification and sequencing of fcDNA with primers �� 1F and �� 1R allowed the identification of paternally-inherited wild-type or mutant alleles in the maternal plasma. In couple 1, paternal SNPs were present at locations rs 10742584 (adenine) and rs 74234654 (adenine) of the ��-globin gene, resulting in a double peak (adenine/thymine and adenine/guanine) at both locations. This result ABT-888 manufacturer indicates that the mutant allele was present in the fetus. Therefore, the fetal genotype can be either a ��-thalassaemia carrier or major for the ��-globin gene mutation at CD41/42. One advantage of this study is that it allows fetal genotype prediction to be carried out for couples in whom both partners are carrying identical mutation. However, if the paternally-inherited mutant allele is present or the paternally-inherited wild-type allele is absent in the fcDNA, the result will still be inconclusive. Nonetheless, the result shows that five of eight couples (more than half) are not at risk of having a homozygous mutant fetus and the screening is more efficient compared to the study carried out by Ding et al.12 This preliminary study will be continued with a larger number of couples to further validate the efficiency. Importance of fetal enrichment This study also demonstrated the importance of the fetal DNA enrichment steps prior to amplification and sequencing of the fcDNA. The fetal DNA enrichment steps were carried out with some modification, described earlier. Initially, the extracted fcDNA was used for direct PCR amplification (primers �� 1F and �� 1R) without undergoing fetal DNA enrichment. Sequencing results revealed the absence of paternally-inherited SNPs at locations rs 10742584 and rs 74234654 in the fcDNA that did not undergo fetal DNA enrichment (figure 3). However, the fcDNA that did undergo fetal DNA enrichment showed double peaks at the mentioned SNP locations. The absence of paternally-inherited SNPs could be due to high background concentration of fcMDNA, which masks the minute concentration of fcFDNA in the maternal plasma. fcMDNA is generally longer than fetal DNA as it is mainly contributed to by lysis of maternal white blood cells. In addition, Kimura et al22 and Chan et al23 showed that the fragment length of fcFDNA extracted from maternal plasma was shorter than 313?bp. Thus, DNA enrichment is an essential step to reduce the concentration of fcMDNA so that the sensitivity of molecular characterisation of fcFDNA can be increased. Figure?3 The importance of the fetal DNA enrichment steps. The double peaks (adenine and thymine) were observed in the enriched free-circulating fetal DNA sample, while only a single peak (thymine) was observed in the non-enriched free-circulating fetal DNA sample. ... As inheritance involves transmission of the haploid genome, the possibility of a fetus inheriting the paternal mutant allele is 50%.