An Improved Method for Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences

Kogan, Scott C.; Doherty, Mark; Gitschier, Jane

doi:10.1097/00006254-198843040-00016

Cited by 114 publications

(140 citation statements)

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“…The DNA of each isolate was then typed by repetitive extragenic palindromic (REP) PCR fingerprinting using the BOX A2R primer (5′-ACG TGG TTT GAA GAG ATT TTC G-3′) (Koeuth et al, 1995). Twenty-five microlitres of PCR reactions contained 5 ml of 5¥ Gitschier Buffer (Kogan et al, 1987), 2.5 ml of 10% dimethyl sulfoxide, 0.4 ml of bovine serum albumin (10 mg ml -1 ), 2.0 ml of 10 mM dNTPs, 1.0 ml of Taq polymerase (5000 u ml -1 ), 10.6 ml of water, 1.5 ml of 10 mM BOXA2R primer; and 2.0 ml of DNA template, containing between 10 and 40 ng -1 ml -1 DNA (Versalovic et al, 1991;Malathum et al, 1998). The PCR programme included (i) initial denaturation at 95°C for 7 min, (ii) 35 cycles of 90°C for 30 s, 40°C for 1 min and 65°C for 8 min, and (iii) final extension at 65°C for 16 min.…”

Section: Enterococcus Genotypingmentioning

confidence: 99%

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Badgley¹,

Thomas²,

Harwood³

2010

Environmental Microbiology

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Enterococcus spp. are utilized worldwide as faecal indicator bacteria, but certain strains exhibit extended survival in environmental habitats and the factors influencing their persistence are poorly understood. We used flowing freshwater mesocosms to explore the effect of submerged aquatic vegetation (SAV) on the persistence of natural enterococci populations from a subtropical lake. The highest mean densities of culturable enterococci over 2 weeks occurred in SAV [8.6 x 10(2) colony-forming units (cfu) per 100 g wet weight], followed by sediments (1.3 x 10(2) cfu per 100 g) and water (18 cfu per 100 ml). However, due to relative differences in the total mass of each substrate in the entire system (water > sediments > SAV), SAV-associated enterococci represented only a minor proportion of the total population. Vegetated mesocosms harboured significantly higher mean cfu per mesocosm and cfu densities in sediments compared with their unvegetated counterparts, suggesting that SAV indirectly facilitates persistence in aquatic habitats. Populations were dominated (> 96%) by a single Enterococcus casseliflavus strain according to BOX-PCR genotyping, which did not change over the 10-month study and strongly suggests bacterial replication in the lake. The presence of such strains in the environment may represent highly competitive, naturalized and reproducing indicator bacteria populations that are not directly related to pollution events.

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Section: Enterococcus Genotypingmentioning

confidence: 99%

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Badgley¹,

Thomas²,

Harwood³

2010

Environmental Microbiology

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“…Haemophiliu (1996). 2, [11][12][13][14][15][16][17] The gels were dried and autoradiographed at room temperature for 2-16 h.…”

Section: Southern Blot Analysis For Factor Viil Gene Inversionsmentioning

confidence: 99%

Current strategy for genetic analysis of haemophilia A families

et al. 1996

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Carrier detection and prenatal diagnosis of haemophilia A, which was based in the last decade mainly on linkage polymorphism analysis, has been greatly facilitated by the recent discovery that two types of inversion disrupting the factor VIII gene are common mutations observed in 42-48% of severe haemophilia A cases. In this study DNA analysis was performed in 64 unrelated severe haemophilia A patients and 173 women belonging to their families, and in four women from a family with a deceased haemophilic relative whose DNA was unavailable (a total of 177 females from 65 unrelated families). Factor VIII gene inversions were found in 32 out of the 65 families (49%), 29 involving recombination with the distal A gene and three with the proximal A gene. Definitive information regarding carriership of haemophilia was provided to all 81 women belonging to the 32 inversion-positive families, among them one woman previously uninformative for any of the polymorphisms examined, five women who were informative only for extragenic polymorphisms, and four suspected carriers who were relatives of the deceased haemophiliac. In 33 inversion-negative families, 96 females were examined by analysis of the BclI restriction fragment length polymorphism (RFLP) in intron 18 and of the multiallelic dinucleotide repeats in introns 13 and 22, followed by analysis of other intragenic polymorphisms. This procedure yielded an informativity rate of almost 100%. Of the 96 females examined by linkage polymorphism analysis, 78 belonged to 25 families with more than one haemophiliac and 29 of them were obligate carriers. In 47 of the 49 suspected carriers linkage polymorphism analysis enabled definition of carriership based on intragenic polymorphisms. 18 of the 96 females belonged to eight families with sporadic haemophilia cases and only eight of the 18 suspected carriers could be diagnosed by exclusion. In nine pregnant women carrying factor VIII gene inversions, mRNA extracted from chorionic villus samples (CVS) was analysed for factor VIII gene inversion by reverse transcription/polymerase chain reaction (RT/PCR). This procedure enabled rapid prenatal diagnoses in six male fetuses. Taken together, our data indicate that a high rate of informativity and carrier definition is possible by the strategy of first screening for factor VIII gene inversions, and, if none are found, sequential use of highly informative intragenic polymorphisms, followed by less informative intragenic and extragenic polymorphisms.

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“…In some cases, the mutation resulted in creation or removal of a predicted restriction enzyme recognition site and in others, the fragment was analysed by direct DNA sequencing. When more than one family was identified with the same point mutation, haplotypes were compared including common polymorphisms in the following restriction enzyme recognition sites: BclI in intron 18 [20], AlwaNI in intron 7, HindIII in intron 19 and and do not include the 19 amino-acid signal peptide. Four subjects with a borderline severely deficient value of FVIII:C = 1% (M824, M826, M833 and M842) are classified as 'severe' based upon their clinical phenotype, as discussed in the text.…”

Section: Mutation Screening and Identificationmentioning

confidence: 99%

Factor VIII mutations in 42 Moldovan haemophilia A families, including 12 that are novel

Sirocova¹,

Tsourea²,

Vicol³

et al. 2009

Haemophilia

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Haemophilia A (HA) is a bleeding disorder caused by mutations within the X-linked F8 gene. A series of 42 unrelated Moldovan patients with HA had their disease-causative mutation determined to provide clinically valuable genotyping information for a historically underserved population and to utilize factor VIII (FVIII) structural information to analyse the effects of haemophilic missense substitutions. DNA samples were analysed to detect intron 22 and intron 1 inversions followed by heteroduplex analysis of PCR-amplified fragments containing all exonic sequences. Missense sites identified by DNA sequencing were visualized in the recently described crystal structures of human FVIII. Of the 26 different point mutations, 12 were novel. Gel electrophoresis identified samples with a second major DNA band that migrated abnormally; these amplified products were sequenced. Thirteen intron 22 inversions and one intron 1 inversion were found. Two patients had large, partial gene deletions and there were six frameshift, two non-sense, two splicing and 16 missense genotypes. Two subjects with an intron 22 inversion and one with a large, partial gene deletion developed an alloimmune inhibitor. Their localization suggests intra- and possibly inter-molecular interactions that are important for the structural integrity and/or procoagulant function of FVIII.

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An Improved Method for Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences

Cited by 114 publications

References 0 publications

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

The effects of submerged aquatic vegetation on the persistence of environmental populations of Enterococcus spp.

Current strategy for genetic analysis of haemophilia A families

Factor VIII mutations in 42 Moldovan haemophilia A families, including 12 that are novel

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