Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma

Objectives To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA). Methods The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples. Results Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series. Conclusions This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA. Copyright © 2007 John Wiley & Sons, Ltd.     

苯甲酸盐厌氧驯化体系中三氯乙烯的还原脱氯特性

采用气相色谱法实时监测了厌氧条件下,以苯甲酸盐为唯一碳源的驯化活性污泥体系对三氯乙烯(TCE)的还原脱氯情况;同时,结合454焦磷酸测序和实时荧光定量PCR技术对该体系中的微生物群落结构及脱卤拟球菌(DHC)的数量进行分析.结果表明,该驯化体系在94 d内能将TCE完全去除,最终转化产物为一氯乙烯(VC),同时伴随大量的甲烷产生;体系中微生物的多样性很高,涵盖了16个门、33个纲、52个目、88个科和129个属,而且体系中约有51.2%的微生物的分类地位尚未明确,说明体系中还存在很多未知的功能菌;体系对TCE的降解过程其实就是还原脱氯菌与其它功能菌相互作用的结果,且起主要还原脱氯作用的是携带tce A功能基因的DHC.     

Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.