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In addition, Li et al10 also showed the feasibility of amplification using peptide-nucleic-acid, which demonstrates high binding affinity to specific sequences for detection of paternally-inherited mutations in ��-thalassaemia. This approach, however, is ineffective when a couple has the same mutation��the paternally and maternally-inherited mutant allele are the same. In situations where a couple has the same mutation, analysis of single nucleotide polymorphism (SNP) can be used to trace the inheritance of mutations as the SNP can be with either of the alleles. The presence of a SNP with the paternal mutant allele or the absence of a SNP with the paternal wild-type allele indicates the inheritance of the paternal mutant allele in the fetus. To the contrary, the presence of a SNP with the paternal wild-type allele or the absence of a SNP with the paternal mutant allele will indicate that the fetus will inherit either one or both wild-type alleles, thus producing a normal or carrier fetal genotype, respectively. For example, Ding et al12 used a SNP marker to detect the presence of a paternally inherited mutant allele in the ��-globin gene and reported that the fetus is not at risk for thalassaemia major when the SNP with the paternally-inherited mutant allele was absent. A simple molecular approach based on allele-specific PCR (AS-PCR) in combination with sequence analysis for haplotype determination on individual samples was reported in previous studies.13�C15 Hence, this study aims to identify SNP located in the flanking regions upstream, adjacent or downstream from the ��-globin gene mutations at CD41/42 (HBB:c.127_130delCTTT), IVS1-5 (HBB:c.92+5G>C) and IVS2-654 (HBB:c.316-197C>T). The results from this study will demonstrate the usefulness of SNP for identification of paternally-inherited alleles in NIPD. Methodology Sample collection Maternal plasma, and parental and CV DNA used in this study were archived clinical samples sent for prenatal diagnosis of ��-thalassemia from 2008 to 2011. PDE4B Ethical approval was obtained from the Medical Ethics Committee of University Malaya Medical Centre (MEC Ref No: 896.8) in accordance with the Declaration of Helsinki. Informed and written consent was obtained from patients before blood collection and CV sampling. DNA from eight couples who had been previously characterised as ��-thalassaemia carriers with ��-globin gene mutations at CD41/42 (couples 1, 2 and 3), IVS1-5 (couples 4, 5 and 6) and IVS2-654 (couples 7 and 8), were used in this study. Maternal plasma was isolated from maternal blood samples by centrifugation at 1900��g for 10?min at 4��C. The plasma was transferred into 1.5?mL microcentrifuge tubes without aspirating the buffy coat layer and recentrifuged at 16?000��g for 10?min at 4��C to remove the cellular nucleic acids.