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PubMed2002

Comparison of conventional techniques to differentiate between Taenia solium and Taenia saginata and an improved polymerase chain reaction-restriction fragment length polymorphism assay using a mitochondrial 12S rDNA fragment.

Given the constraints of classical diagnostic methods, i.e., morphological and isoenzymatic studies of proglottids, a polymerase chain reaction test complemented with restriction enzyme analysis has been modified by redesigning one of the primers to reduce nonspecific amplifications experienced when using field samples. The use of these new, highly cestode-specific primers and the restriction enzyme Ddel led to the development of a diagnostic assay that clearly distinguishes between Taenia saginata and T. solium proglottids in field samples. This assay confirms the presence of T. saginata in Ecuador. DNA amplification of some of these taeniids showed different patterns, suggesting the possibility that strain differences exist. These results demonstrate the need for development of useful molecular assays as reliable tools for epidemiological studies on cestodes.

Animals↗
PubMed1986

Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency.

DNAs from unrelated healthy individuals and unrelated individuals affected with 21-hydroxylase deficiency (congenital and late-onset adrenal hyperplasia) were digested with seven restriction enzymes and hybridized with a cDNA probe specific for human 21-hydroxylase genes. Associations were found between restriction fragments and the two forms of the disease: The late onset form is associated with a double dose of a 14 kb fragment generated by EcoRI and with a triple dose of a 3.2 kb fragment generated by Taq I in patients with HLA B14 haplotypes; The classical congenital form is negatively associated with the 14 kb fragment and with a 3.7 kb fragment generated by Taq I in patients with HLA Bw47 haplotypes. A 3.2 kb Taq I fragment is negatively associated with the HLA B8 haplotypes. The other five enzymes tested give no polymorphisms or polymorphisms without correlation with the two forms of the disease.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

Animals↗
PubMed1985

Segregation of restriction fragment length polymorphism in an interspecies cross of laboratory and wild mice indicates tight linkage of the murine IFN-beta gene to the murine IFN-alpha genes.

Southern blot analysis with murine (Mu) interferon (IFN)-alpha cDNA of restricted genomic DNA of three inbred strains of mice belonging to the species Mus musculus domesticus (BALB/c, C57BL/6, and DBA/2) revealed only a limited degree of polymorphism. For example, with HindIII there were only two polymorphic bands out of 14 hybridizing fragments. With Mu IFN-beta cDNA there was no polymorphism at all between BALB/c and C57BL/6 in DNA restricted with seven different enzymes. In contrast, HindIII-restricted DNA of an inbred strain of wild mice (M. spretus Lataste) hybridized with the IFN-alpha probe displayed a high degree of polymorphism compared with the three strains of laboratory mice and was also polymorphic when probed with IFN-beta cDNA. Although M. musculus domesticus and M. spretus Lataste represent different species, certain interspecies crosses are possible in the laboratory. This enabled us to follow segregation of restriction fragment length polymorphism in HindIII-restricted DNA obtained from 18 backcross progeny of a (DBA/2 X M. spretus)F1 X DBA/2 interspecies cross. There was complete coincidence between the segregation of parental (DBA/2) and (DBA/2 X M. spretus)F1-type IFN-beta and IFN-alpha restriction fragment length polymorphism, indicating tight linkage of the IFN-beta and IFN-alpha genes. In addition, in 15 of 18 progeny the segregation coincided with that of the brown locus on chromosome 4, in accord with previous results obtained with the IFN-alpha probe in strains derived from crosses between BALB/c and C57BL/6 mice. Thus, the Mu IFN-beta gene is tightly linked to the Mu IFN-alpha gene cluster on chromosome 4 near the brown locus.

Animals↗