Identification of foetal DNA in maternal plasma using semi–nested PCR: A possible approach for non–invasive prenatal diagnosis

Abstract

IntroductionPrenatal diagnosis using foetal DNA is essential for detection of the foetal genotype in life threatening genetic disorders such as thalassaemia. Individuals with β-thalassaemia major require regular blood transfusions for survival. Epigenetic studies have revealed differences in methylation status in CpG sites between foetal and maternal DNA. The objectives of this study are to identify informative CpG sites in the β-globin gene and develop a semi-nested methylation-specific PCR for amplification of chorionic villi (CV) DNA (trophoblasts that contribute to foetal DNA). MethodsExtracted DNA from 15 maternal blood and their respective CV samples were subjected to bisulfite conversion. The β-globin gene containing three selected CpG sites and the β-gene mutation at codon 41/42 were amplified using a pair of primers (F1 and R1). Amplicons were sequenced and the methylation status of each CpG site of the converted maternal (cM) was compared to the converted CV (cCV) DNA. Three rounds of semi-nested PCR using three methylation-specific reverse primers (R2, R3 and R4) were amplified in succession with a common forward primer (F1). Results & DiscussionSequencing results confirmed the three CpG sites were hypermethylated in cM DNA and only partially methylated in cCV DNA. The semi-nested PCR showed amplification in 11 cCV DNA while no band was observed in all maternal DNA. The sensitivity and specificity of this approach is 73% and 100% respectively. ConclusionThree rounds of semi-nested PCR on cM and cCV DNA have allowed the identification of circulating foetal DNA in maternal plasma. This technique may be utilised for non-invasive prenatal diagnosis of specific β-globin gene mutations from foetal DNA in maternal plasma.