Establishment Of New Detection Techniques For Fetal Paternal Point Mutations In Maternal Plasma And Their Significance In Noninvasive Prenatal Diagnosis

Background and purposes: Free fetal DNA has been found in maternal plasma recently, which facilitates the development of noninvasive prenatal diagnostic approaches based simply on the analysis of a maternal blood sample.The detection of the paternally transmitted pathogenic allele in maternal plasma has been reported to be applicable to the prenatal diagnosis of paternally inherited autosomal dominant traits. If a similar approach is used for the prenatal assessment of autosomal recessive conditions, an invasive prenatal diagnostic procedure would be avoided in 50% of these pregnancies, in whom the lack of inheritance of the paternal mutation by the fetus is confirmed by maternal plasma analysis. However, the low quantity of fetal DNA in the maternal circulation and interference from an excessive amount of maternal DNA makes detection of fetal mutations difficult. The diagnostic reliability depends on the absolute sensitivity and specificity of the assay system. Present clinical DNA diagnosis mainly concentrates on detection of point mutation and single-nucleotide polymorphism (SNP). The technique detecting the discrimination of single-nucleotide differences between fetal DNA and maternal DNA is challenging and demands the adoption of more highly sensitive and specific analytical systems. However, reported diagnostic procedures are still beyond our satisfaction. DNA ligase has the capacity to accurately discriminate a single nucleotide polymorphism. Two adjacent oligonucleotide primers hybridized to a single strand of target DNA will be ligated by ligase only if there is an exact match to the target sequence. Ligation will not occur in the presence of a mismatch. The fidelity of DNA ligases proviedes the basis for developing ligase-based technologies to highly sensitively detect low abundance point mutations. Gap-gap ligase chain reaction (gap-LCR) and ligase detection reaction (LDR) achieve both signal detection and amplification through repeated strand reactions catalyzed by DNA ligase. The aim of the study is to detect low abundance point mutations by using Gap-LCR and real-time PCR and...