Rapid Prenatal Diagnosis Of Chromosomal Aneuploidy Using Real-time Multiplex Ligation-dependent Probe Amplification (Real-time MLPA)

Chromosomal aneuploidy is a kind of genetic disease which has high incidence, severe symptom and so far, can not be cured. Rapid and accurate prenatal diagnosis of chromosomal aneuploidy with high throughput is very meaningful in reducing the birthrate of suffering fetus, and increasing the whole quality of population. This dissertation describes a novel technology namely real-time multiplex ligation-dependent probe amplification which aimed at the rapid and accurate prenatal diagnosis of chromosomal aneuploidy.In chapter one, the status of the prenatal diagnosis of chromosomal aneuploidy was reviewed. Traditional cytogenetic method and molecular methods were summarized briefly and the concept of real-time multiplex ligation-dependent probe amplification was introduced.In chapter two, principle and design of real-time multiplex ligation-dependent probe amplification as well as the detection system and data analysis of this technique were described. The influence of the reaction factors on the hybridization reaction and real-time PCR reaction were examined, then the optimized reaction system was established. Two data analysis methods based on different theory were described, with which we could scan the raw output bidirectionally and rendering the diagnostic method more reproducible and reliable.In chapter three, real-time multiplex ligation-dependent probe amplification was validated using case-control study. Clinical samples included 32 cases of trisomy 21, 11 cases of trisomy 18, 1 case of trisomy 13 and 100 samples of normal control. The results showed that all trisomy cases were differentiated distinctly from the normal controls. It was further observed that 1.2 times change in chromosome dosage could be detected by this technique, thus it would be expected that detection of mosaicism as well as noninvasive prenatal diagnosis could be performed.