First-trimester prenatal diagnosis of cystic fibrosis using the polymerase chain reaction: Report of eight cases

Eight pregnancies at risk for cystic fibrosis have been monitored by first-trimester prenatal diagnosis with DNA amplification analysis. The polymerase chain reaction (PCR) was used in all cases to amplify the region detected by KM 19. In two cases, the region detected by CS·7, another DNA probe tightly linked to the CF locus, was also examined. The results of the PCR determinations were confirmed using the Southern blotting procedure, by segregation analysis of restriction fragment length polymorphisms (RFLPs) relative to XV-2c, J3·11, metH, metD, and KM19 probes.     

Prenatal diagnosis of autosomal dominant polycystic kidney disease using flanking DNA markers and the polymerase chain reaction

A prenatal diagnosis was carried out on a 9-week-old fetus at risk for autosomal dominant polycystic kidney disease (ADPKD). Ten members of the family were previously typed using five DNA markers linked to the PKD1 locus on chromosome 16, and one marker linked to the putative PKD2 locus on chromosome 2. The polymerase chain reaction (PCR) was used to amplify the D16S125 locus. Pairwise and multipoint lod scores indicated that the family was most likely segregating a PKD1 mutation. The fetus inherited the disease haplotype from the affected parent. Diagnostic accuracy was greater than 99 per cent, taking into account the possibility of genetic heterogeneity.     

In vitro amplification of the α1-antitrypsin gene: Application to prenatal diagnosis

Discrimination of the M, Z, and S alleles of α₁-antritrypsin (AAT) has been carried out using in vitro gene amplification with the polymerase chain reaction (PCR). Amplification of 90 nucleotides surrounding the Z mutation site and 120 nucleotides surrounding the S mutation site dramatically improves the sensitivity and reliability of allele-specific oligonucleotide (ASO) hybridization for direct detection of these alleles. Analysis is performed using Southern blots or dot blots hybridized with 19 base oligonucleotides and differentially washed for allele specificity. Amplification of the Z and S mutation sites can be combined in one PCR to allow detection of both mutations when analysed by gel electrophoresis and Southern transfer. This technique can be performed reliably using less than 0·1 μg of genomic DNA or less than 100 amniocytes or white blood cells. This technique has been used to perform prenatal diagnosis on a chorionic villus sample (CVS) in a fetus at risk for the ZZ Pi type form of AAT deficiency.     

Identification of a case of maternal uniparental disomy of chromosome 10 associated with confined placental mosaicism

We report a case of maternal uniparental disomy of chromosome 10 discovered after chorionic villus sampling (CVS). Direct preparations revealed mosaic trisomy 10, while cultured CVS cells, as well as amniotic fluid cells, showed only a normal 46,XY complement. DNA analysis using microsatellite markers showed both chromosomes 10 to have been inherited from the mother. The pregnancy was complicated by polyhydramnios. A phenotypically normal male infant of appropriate size was delivered by Caesarean section at 41 weeks' gestation. Since only the direct preparations showed trisomy 10, this case illustrates the importance of CVS direct preparations in the detection of pregnancies at risk of uniparental disomy (UPD). Although the increased frequency of confined placental mosaicism (CPM) diagnosed when direct preparations are performed has been viewed negatively, identification of both CPM and UPD may have biological and clinical significance for a pregnancy. Even though only a single case of maternal disomy 10 is reported here, the apparently normal phenotype provides evidence that there are no major imprinted loci on chromosome 10 that affect in utero growth and development. However, other potential effects such as mental retardation will require long-term follow-up of this as well as additional cases.     

First-trimester prenatal diagnosis of osteogenesis imperfecta type II by DNA analysis and sonography

Osteogenesis imperfecta type II was diagnosed prenatally by analysis of DNA obtained from chorionic villus sampling (CVS) performed at 12 weeks of gestation in a woman who previously had had an affected child. The father had been shown to be mosaic for a mutation in the gene (COL1A2) which encodes the α2(I) chain of type I collagen. An affected fetus was predicted by detection of the mutation in amplified chorionic villus genomic DNA. Ultrasound examination at 13 weeks 4 days demonstrated femoral deformity and virtual absence of calvarial mineralization. In pregnancies at risk for osteogenesis imperfecta type II, sonographic evidence of skeletal abnormalities may be evident by 13 weeks' gestation.     

Prenatal diagnosis of fragile x syndrome: (cgg)n expansion and methylation of chorionic villus samples

Fragile X syndrome is the most common form of inherited mental retardation, due to an expansion of the (CGG)_n trinucleotide repeat in the FMR-1 gene and hypermethylation of its 5′ upstream CpG island. Two major problems remain to be resolved for fragile X prenatal diagnosis: the abnormal methylation patterns of chorionic villus samples (CVS) and the inability to predict the mental status of females with the full mutation. We present here the results of ten prenatal diagnoses of fragile X syndrome using Southern blotting and polymerase chain reaction (PCR) amplification, and the analysis of 50 further CVS to test the methylation status of the CpG island of the FMR-1 gene. In the ten ‘at-risk’ CVS, eight normal (five males and three females) and two affected male fetuses were detected. Absence of methylation in the CVS was observed in two cases, which was not found upon subsequent examination of the newborn or of fetal tissues. In the 50 CVS not ‘at risk’ for fragile X syndrome, abnormal fragment patterns for probe StB12.3 were detected in 32 per cent for female and 24 per cent for male fetuses. This abnormal pattern could be due to absent or partial methylation of the CpG island of the FMR-1 gene in chorionic villus tissues.     

富勒烯对PCR反应的抑制作用

以110bp单链DNA为模板,研究富勒烯(C60)对聚合酶链式反应(polymerase chain reaction,PCR)的影响.实验结果表明,随着C60浓度的增加,PCR反应被显著抑制;将Taq DNA聚合酶、单链DNA模板与C60孵育后,其PCR扩增产物均显著减少;增加PCR反应体系中的Taq DNA聚合酶量,可消除C60的抑制作用,但增加起始单链DNA模板的数量,效应不明显,上述研究结果说明,C60不仅可抑制Taq DNA聚合酶活性,同时对DNA模板也具有一定的损伤作用.     

Prenatal diagnosis of haemoglobin disorders by cordocentesis at 12 weeks' gestation

Prenatal diagnosis of haemoglobin disorders is accepted to be a useful procedure to avoid births of infants with homozygous diseases. Advances in sampling and molecular techniques, such as polymerase chain reaction (PCR) and chorionic villus sampling (CVS), have made earlier and safer first-trimester prenatal diagnosis possible. However, these procedures need previous studies of at-risk couples, which can be very time-consuming when a number of different β-thalassaemia mutations occur in the region. We describe the possibility of making a first-trimester prenatal diagnosis by cordocentesis and fetal blood analysis at the 12th week of gestation. We found no statistically significant difference (p>0.05) between β/γ values in fetuses at the 12th and 18th weeks of gestation. In seven affected fetuses aborted at the 12th week of gestation, the diagnosis was confirmed in all cases by PCR analysis. These findings suggest that early cordocentesis could be an alternative procedure to CVS and PCR analysis.     

长牡蛎尼氏单孢子虫的初步研究

采集大连大窑湾养殖区的长牡蛎(Crassostrea gigas),抽取其血淋巴液在显微镜下观察,观察到尼氏单孢子虫(Hap-losporidium nelsoni)原生质体的类似物.对抽取的血淋巴液进行总基因组DNA的提取,将扩增单孢子虫(Haplosporidiosis)SSUrDNA区域的引物对HAP-F2和HAP-R2进行改进,经过特异性试验证明其特异性很好,得到引物对HAP-F和HAP-R,应用此对引物进行聚合酶链式反应(PCR),结果扩增出239bp条带.将PCR产物进行测序,经序列比对分析确定为尼氏单孢子虫.同时,采用尼氏单孢子虫的特异性DNA探针MSX1347对抽取的长牡蛎血淋巴液进行原位杂交,结果也检测到了尼氏单孢子虫.     

Analysis of parental strain DNA fragments existing in GEMs-Fhhh

There were 6 target DNA fragments of the three parental strains existing in the cell of GEMs( genetically engineered microorganism strain) Fhhh measured in this research by PCR(polymerase chain reaction). The determination showed that GEMs Fhhh contained all the 6 target DNA fragments, mnpl, mnp2, lipl, lip2, FLOI and 16S rDNA, and had the molecular genetic stability. Meanwhile the PCR production of each parental strain could only had its target DNA fragments and was different from each other. It may illustrate that the technique of the inter-kingdom protoplast fusion for the construction of GEMs Fhhh through the process of intercellular gene recombination could be used as a reliable bioengineefing technique to create the soecific functional stain for the nollution control.     

Prenatal diagnosis of DHPR deficiency by direct detection of mutation

Prenatal diagnosis was requested by a family carrying a 3 base-pair insertion in the dihydropteridine reductase (DHPR) coding region. A chorionic villus sample was obtained and fetal DNA was isolated directly from this. Diagnosis was performed by a polymerase chain reaction (PCR)-based technique, with a simple electrophoretic assay for the insertion. The fetus was found to be heterozygous for the insertion. This is the first time that prenatal diagnosis of DHPR deficiency has been performed by direct detection of the mutation.     

Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders

We report our experience of 14 preimplantation genetic diagnosis (PGD) cycles in eight couples carrying five different single gene disorders, during the last 18 months. Diagnoses were performed for myotonic dystrophy (DM), cystic fibrosis (CF) [ΔF508 and exon 4 (621+1 G>T)], fragile X and CF simultaneously, and two disorders for which PGD had not been previously attempted, namely neurofibromatosis type 2 (NF2) and Crouzon syndrome. Diagnoses for single gene disorders were carried out on ideally two blastomeres biopsied from Day 3 embryos. A highly polymorphic marker was included in each diagnosis to control against contamination. For the dominant disorders, where possible, linked polymorphisms provided an additional means of determining the genotype of the embryo hence reducing the risk of misdiagnosis due to allele dropout (ADO). Multiplex fluorescent polymerase chain reaction (F-PCR) was used in all cases, followed by fragment analysis and/or single-stranded conformation polymorphism (SSCP) for genotyping. Embryo transfer was performed in 13 cycles resulting in one biochemical pregnancy for CF, three normal deliveries (a twin and a singleton) and one early miscarriage for DM and a singleton for Crouzon syndrome. In each case the untransferred embryos were used to confirm the diagnoses performed on the biopsied cells. The results were concordant in all cases. The inclusion of a polymorphic marker allowed the detection of extraneous DNA contamination in two cells from one case. Knowing the genotype of the contaminating DNA allowed its origin to be traced. All five pregnancies were obtained from embryos in which two blastomeres were biopsied for the diagnosis. Our data demonstrate the successful strategy of using multiplex PCR to simultaneously amplify the mutation site and a polymorphic locus, fluorescent PCR technology to achieve greater sensitivity, and two-cell biopsy to increase the efficiency and success of diagnoses. Copyright © 2002 John Wiley & Sons, Ltd.     

SO2诱导驯化城市污水处理厂氧化沟微生物的多样性分析

对城市污水处理厂氧化沟采集的微生物用低浓度SO2诱导驯化6d以上可培养出脱SO2微生物种群,它们之间相互协同,共同行使着对溶于水中的SO2生化降解的生物学功能,对SO2的生化降解速率高达888mg·（L·h）^-1,1．5h水中的SO3^2-的降解效率〉85％．脱硫菌样品经DNA提取及16SrRNA基因片段的聚合酶链反...     

水中轮状病毒实时定量PCR外标准品的构建

采用细胞培养和T-A克隆技术,在轮状病毒主要结构蛋白VP7基因序列上设计合成引物,经PCR扩增后将特异性产物连接入pGEM-T-easy载体中,经过酶切鉴定和测序分析获得轮状病毒cDNA标准品.利用常规PCR和实时定量PCR方法对所获得的cDNA标准品进行特异性、稳定性和重复性指标的检验.结果表明,利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系(斜率为-3.353,R2=0.995);实时定量PCR熔解曲线分析表明,温度在81℃±0.5℃的PCR产物是轮状病毒VP7序列上的特异性产物,表明此标准品是轮状病毒特异性的;同时,所制备的标准品在实时定量PCR检测中具有较大的线性范围(9×100～9×1011 copies/μL,每个反应最低可以检测到9个拷贝数的轮状病毒cDNA,表明利用该标准品进行实时定量PCR分析时具有较高的检测灵敏性;此外,该标准品具有较高的稳定性和重复性(3次独立实验CV值在0.2%～0.9%之间),可长期稳定保存.构建的标准品可用作检测水环境中轮状病毒的实时荧光定量PCR的外标准品.     

Dynamic changes of microbial community diversity in a photohydrogen producing reactor monitored by PCR-DGGE

A PCR-DGGE (denaturing gradient gel electrophoresis of polymerase chain reaction) protocol was used for monitoring the dynamic changes in the microbial population during photohydrogen production. Total DNA was extracted directly from the mixed bacterial community in the reactor and subjected to PCR with V3-16S rDNA and pufM gene primers, and the amplifications were then analyzed by DGGE. The DGGE patterns demonstrated the dynamics of community structure and the shift of microbial diversity, which correspond...     

Resolution of DNA linkage discrepancies through analysis of a VNTR locus in a family study of cystic fibrosis

First-trimester prenatal diagnosis of a fetus at 25 per cent risk for cystic fibrosis (CF) was performed by indirect linkage analysis of polymorphic markers using Southern blotting and polymerase chain reaction (PCR) amplification. The results revealed discrepancies in the allelic patterns between the father and the affected child, thereby complicating the prediction of fetal outcome. Analysis of a highly polymorphic VNTR locus within the human retinoblas-toma (RB) gene on chromosome 13 showed that the affected child and the fetus did not have the same biological father, and therefore the affected child could not be used to determine linkage of markers in the father of the fetus. The analysis of VNTR loci can be an effective method of resolving conflicting data during prenatal diagnosis of monogenic diseases.     

Verification of carrier status for Becker muscular dystrophy from analysis of a blighted ovum

The polymerase chain reaction (PCR) was used on material from a blighted ovum to confirm indirectly the carrier status of a woman with a family history of Becker muscular dystrophy. Conventional testing including creatine kinase levels, muscle biopsy, and EMG had been inconclusive, and on the basis of one elevated creatine kinase level, the woman had been designated a possible carrier. Ultrasound examination at 10 weeks of pregnancy indicated a blighted ovum, from which DNA was subsequently extracted and subjected to PCR testing for determination of sex and genotypic status with respect to the known familial deletion of the dystrophin gene. The blighted ovum was found to have a Y chromosome and also to be deleted for at least exon 6 of the dystrophin gene, indirectly indicating that the mother most likely carried the family mutation for Becker muscular dystrophy.     

Prenatal analysis of the insulin receptor gene in a family with leprechaunism

We report on the prenatal diagnosis of a fetus at risk of leprechaunism. We had previously determined the nature of the causative mutation in the insulin receptor gene in this family. The mutation removes a restriction site for the enzyme Mbo II. Genomic DNA was extracted from a chorionic villus sample and the 3′ half of exon 2 was amplified by the polymerase chain reaction (PCR) followed by restriction digest. Using this method, we correctly predicted an unaffected child.