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Biochemical and clinical diagnostic aspects of circulating nucleic acids

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Disciplines
  • Biology
  • Design

Abstract

We investigated the size distribution of fetal DNA using a combination of agorose gel electrophoresis, Southern blot and real time PCR assay. Our results showed plasma DNA presents apoptotic characteristics and the cell free fetal DNA is generally smaller than maternal plasma DNA. Therefore, the fetal DNA can be enriched by size separation. The enriched fetal DNA in turn can be used for the detection of paternally inherited STR sequence that is masked by maternal sequences. Next, we examined paternally inherited single gene point mutations in FGFR3 gene and β-globin gene respectively. Our analysis showed we could be able to detect the fetal single gene mutations in maternal plasma by using size separation. These studies indicated the size-fractionation of plasma DNA is very useful for non-invasive prenatal determination of fetal point mutations. Several reports indicated that cell free fetal DAN can be detected in the urine of pregnant women. We attempted to reproduce those data. We examined the urinary DNA from normal pregnant women, as well as from pregnancies complicated by HELLP syndrome. In no instance were we able to detect fetal DNA in maternal urine. Our data suggest cell free fetal DNA is not readily detectable in maternal urine. We also examined paternal RhD genotype by real time PCR assay since it is important to counsel a couple about the risk of HDN. We tested 39 samples obtained from males who had been serologically typed to be RhD. We designed two multiplex real-time quantitative PCR assays to simulataneously detect the RhD gene in relation to the SRY sequence and the GAPDH sequence, respectively. Our study showed the two assays were in complete concordance. Therefore, the result indicated that real time PCR can potentially be used for the determination of RhD zygosity.

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