Molecular prenatal diagnosis: the impact of modern technologies

Originally prenatal diagnosis was confined to the diagnosis of metabolic disorders and depended on assaying enzyme levels in amniotic fluid. With the development of recombinant DNA technology, molecular diagnosis became possible for some genetic conditions late in the 1970s. Here we briefly review the history of molecular prenatal diagnostic testing, using Duchenne muscular dystrophy as an example, and describe how over the last 30 years we have moved from offering testing to a few affected individuals using techniques, such as Southern blotting to identify deletions, to more rapid and accurate PCR-based testing which identifies the precise change in dystrophin for a greater number of families. We discuss the potential for safer, earlier prenatal genetic diagnosis using cell free fetal DNA in maternal blood before concluding by speculating on how more recent techniques, such as next generation sequencing, might further impact on the potential for molecular prenatal testing. Progress is not without its challenges, and as cytogenetics and molecular genetics begin to unite into one, we foresee the main challenge will not be in identifying the genetic change, but rather in interpreting its significance, particularly in the prenatal setting where we frequently have no phenotype on which to base interpretation. Copyright © 2010 John Wiley & Sons, Ltd.     

阴离子粘土(LDH)对DNA吸附行为的研究

以4种阴离子粘土做吸附剂,研究了阴离子粘土对DNA的吸附行为.同时,采用XRD、FTIR、UV-vis等表征手段对吸附前后的材料进行研究.吸附结果显示,二元阴离子粘土对DNA的吸附量高于三元阴离子粘土;3:1型阴离子粘土对DNA吸附力强于2:1型阴离子粘土.4种材料对DNA的吸附均符合Langmuir、Freundlich两种吸附等温模型,且Langmuir吸附等温模型拟合度更高,说明阴离子粘土对DNA的吸附为单层吸附.XRD结果显示,吸附前后阴离子粘土基本结构并未发生改变,晶形完好,层间距未有明显变化,表明阴离子粘土对DNA的吸附仅发生在表面,DNA并未进入阴离子粘土层间结构中.UV-vis及电泳结果显示,吸附前后DNA的构型并未发生改变,阴离子粘土的吸附并未对DNA产生较大的影响.     

彗星试验检测酚类化合物对小鼠脾脏细胞DNA损伤的研究

应用彗星试验技术,研究了酚类化合物对小鼠脾脏细胞DNA的损伤作用,试验结果表明,12种酚类化合物对小鼠脾脏细胞均能引起不同程度的DNA损伤,并呈现剂量效应关系.与对照组相比,均有显著性差异(P＜0 05,P＜0 01).试验结果亦表明,其DNA的损伤程度与化学结构有一定的关系.      

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19–25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal β^E-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 µL of plasma from pregnant women whose husbands carried Hb E. The β^E-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal β^E-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal β^E-globin gene should prove useful in a prevention and control program of Hb E/β-thalassemia in countries where the β^E-globin gene is prevalent. Copyright © 2003 John Wiley & Sons, Ltd.     

长江中下游环境DNA宏条形码生物多样性检测技术初步研究

长江生物多样性在人为影响下面临严重威胁,物种监测是生物多样性保护的基础,为完善长江水生态监测体系,实现高效无损伤的物种监测,在长江中下游干流3个江段（新滩、安庆和芜湖）采集水样,建立长江水样环境DNA宏条形码物种检测体系并评估其有效性.结果表明:①长江中下游环境DNA宏条形码检测到32个物种,包括20种鱼类、1种水生哺乳动物（长江江豚）和11种陆生动物,其中鱼类物种包括鲤形目、鲇形目、鲈形目和鲱形目,其种数占鱼类总种数的比例分别为60%、25%、10%和5%.②长江中下游渔获物中资源量居首位的鲤形目在环境DNA调查中序列数最多,占鱼类总序列的96.2%,其次为鲱形目（占比为3.5%）,鲇形目和鲈形目占比较低,分别为0.2%和0.1%,4个类目序列相对丰度与渔获物种资源量组成差异较大.③环境DNA调查次数约占传统渔获物调查次数的几十至几百分之一,采样时间不足努力量最少的渔获物调查的1%,检测到的鱼类种数为传统调查总数的31%~49%.④安庆采样点位于长江中下游长江江豚密度最高的江段,其环境DNA检出率和序列相对丰度在3个采样点中均最高.研究显示:长江水样环境DNA包含水陆复合生态系统的生物多样性信息,利用水样环境DNA宏条形码可检测不同类群的水生和陆生物种;对于鱼类物种检测,环境DNA宏条形码比传统调查方法效率更高,可对传统调查结果进行补充;环境DNA宏条形码生物多样性检测主要受分子标记体系和核酸序列数据库限制,获取全面的物种多样性和资源量信息需要对检测分析方法进行进一步完善.      

分层型水库藻类垂向演替的水质与细菌种群调控

浮游藻类和细菌是水生食物网的重要组成部分,在水源水库生态系统物质循环和能量流动方面发挥关键作用,藻类的空间演替与水体细菌种群代谢和结构演变关系密切.因此,本文以李家河水库为研究对象,在分析水库水质指标基础上,采用高通量DNA测序技术和Biolog技术,研究水库藻类暴发期,浮游藻类和细菌群落结构垂向演替特征及其与水质的偶联关系.结果表明,李家河水库在8月处于热分层期,水体pH、DO和NH~+_4-N随深度变化均逐渐降低(P0.001).藻密度和Chla呈现同步变化趋势(P0.001),表层最大分别为3 364.33×10~4 cells·L~(-1)和7.03μg·L~(-1).藻类群落结构在水深0 m和3 m处以微囊藻为主,而在水深6 m处,小环藻取代微囊藻成为最优藻属,相对丰度达57.28%.Biolog结果表明,微囊藻的暴发对细菌代谢活性及其相对丰度产生较大影响,但细菌种群代谢活性多样性变化不显著.高通量测序共发现1 420个OTUs,隶属于10个细菌门类,其中放线菌门(Actinobacteria)和变形菌门(Proteobacteria)在不同水层均为最优门类,其相对丰度之和达50%以上;绿菌门(Chlorobi)和浮霉菌门(Planctomycetes)的相对丰度随着水深增加发生显著变化,均在6 m深度达到最大值,分别为10.29%和6.78%,且与水层藻密度呈显著负相关(P0.05);厚壁菌门(Firmicutes)和芽单胞菌门(Gemmatimonadetes)与藻密度呈显著正相关(P0.05).热图(Heat map)指纹图谱表明,李家河水库的细菌群落结构垂向分布差异显著,且随着水深的增加,细菌群落分布更均匀并趋于多样性.冗余分析(RDA)表明,细菌和藻类的群落结构的垂向分布受不同水质指标调控且差异显著.对水源水库藻类暴发期藻类与细菌群落的偶联机制进行探究,为水源藻华消涨的分子微生态驱动机制研究提供科学依据.     

单细胞凝胶电泳技术在环境污染物检测中的应用

主要介绍了单细胞凝胶电泳技术(SCGE),包括SCGE的原理、方法及其在环境污染物检测中的应用,该技术可用多种污染物的检测且灵敏度高,具有广阔的应用前景。即SCGE对大气污染物、重金属、醛类及辐射污染的检测,具有高度敏感性,是具有广阔应用前景的新技术。