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Molecular phylogenetic relationships of puffer fish inferred from partial sequences of cytochrome b gene and restriction fragment length polymorphism analysis.

Phylogenetic relationships among puffer fish were investigated by comparing cytochrome b gene sequences and restriction endonuclease assays of 16 species from Taiwan. DNA was prepared for sequencing by PCR. No variation in sequences was detected among individuals within each species. Direct estimates of mitochondrial cytochrome b gene sequence divergence among 16 puffer fish were from 3.41 to 31.78%. Different restriction patterns were found among 16 puffer fish with 10 restriction endonucleases, whereas no variation in patterns was detected among individuals within each species. The polymorphisms obtained by RFLP have provided a new set of genetic markers for the accurate identification of sibling puffer species. It is the first molecularly based study of puffer diversity and sheds light on the evolution and taxonomy of this major puffer fish family.

Animals↗

Determination of complement factor H functional polymorphisms (V62I, Y402H, and E936D) using sequence-specific primer PCR and restriction fragment length polymorphisms.

BACKGROUND: Complement factor H (CFH; HF) is an essential regulatory protein that plays a critical role in the homeostasis of the complement system in plasma. Several polymorphisms and mutations in the complement factor H gene (CFH; HF1) have been identified. These have revealed interesting associations with hemolytic-uremic syndrome and age-related macular degeneration. METHODS AND RESULTS: The aim of this study was to develop a rapid and reliable assay for determining genotypic variants of the CFH gene. Sequence-specific primer PCR and restriction fragment length polymorphism techniques were chosen for the analysis of CFH polymorphisms. The assays detected the following published single nucleotide polymorphisms of CFH in our Caucasian population (n = 271): rs800292, 257G-->A (V62I); rs1061170, 1277T-->C (Y402H); and rs1065489, 2881G-->T (E936D). The allele frequencies (257G = 0.850, 1277T = 0.574, and 2881G = 0.839) that we obtained from a healthy Hungarian population were consistent with previously published results. CONCLUSION: These analytical methods are simple, reliable, and rapid to perform, and are amenable to automation. Therefore, they could facilitate large-scale genotypic analyses of the CFH gene in various diseases, such as hemolytic-uremic syndrome, age-related macular degeneration, and cardiovascular diseases.

Adult↗

Genomic variation of aquatic birnaviruses analyzed with restriction fragment length polymorphisms.

Aquatic birnaviruses are the most ubiquitous and diverse group of viruses in the family Birnaviridae. Several cause different diseases in a variety of fish species, such as infectious pancreatic necrosis virus in salmonids in North America, Europe, and Asia and European eel virus in eel in Asia. Most isolates are antigenically related and belong to a single serogroup (serogroup A) comprising nine serotypes. Previous studies with monoclonal antibodies have demonstrated considerable variation in epitope profiles even among strains within a single serotype. The few studies of genomic variation among these viruses, which have focused on the NS/VP3 coding region, demonstrated the existence of several genogroups that generally did not correlate with antigenic groups. In this study, PCR was used to amplify a 1,180-bp cDNA genomic fragment representing most of the VP2 (the major outer capsid protein) coding region from five serotype A type strains and 17 Asian isolates. The PCR products were digested with nine different restriction enzymes. Restriction fragment length polymorphism profiles demonstrated heterogeneity among the tested viruses; however, the isolates from Asia were closely related to each other. Cluster analysis of the restriction fragment length polymorphism patterns demonstrated that these viruses could be divided into four major genogroups. In contrast to previous studies of variation in the NS/VP3 coding region, these genogroups based on variation in the VP2 coding region correlated with a serological classification based on VP2-specific monoclonal antibody reaction patterns. Furthermore, all Asian isolates tested belonged to one genogroup typified by the serotype type strain Ab.

Animals↗

Homogeneous restriction fragment length polymorphism analysis of the ribosomal DNA repeating unit in New World Leishmania.

We have studied the Sau 3AI restriction length polymorphisms (RFLP) of the non-transcribed ribosomal spacer of Leishmania isolates from the mexicana and braziliensis complexes, using cloned sequences of Leishmania garnhami and Leishmania braziliensis. The L. garnhami probe produced very complex but conserved patterns in the homologous organisms, and these were shared by all the mexicana complex isolates at intermediate stringency conditions. The small subunit rRNA coding region within the probe also revealed a polymorphic Sau 3AI site exclusive of the braziliensis isolates. The braziliensis probe, containing only spacer sequences, yielded simple and very homogeneous patterns in all braziliensis isolates regardless of their geographical origin. Two main groups are identified in the New World isolates by the RFLP analysis in coincidence with the accepted mexicana and braziliensis complexes.

Animals↗

From the chromosome to DNA: Restriction fragment length polymorphism analysis and its clinical application.

Understanding how chromosomal alterations contribute to acquired and inherited human disease requires the ability to manage the enormous physical and informational complexity of the deoxyribonucleic acid (DNA) packaged within. Important concepts and techniques involved in the analysis of DNA include restriction enzymes, Southern blotting, and restriction fragment length polymorphism/linkage analysis. These techniques have been essential in the understanding and diagnosis of several syndromes associated with the head and neck. The purpose of this article is to introduce DNA structure, describe some techniques fundamental to DNA analysis, and provide a brief overview of the clinical applications of this technology with respect to dentinogenesis imperfecta and oral field cancerization.

Blotting, Southern↗

Application of silver staining to the rapid typing of the polymorphism of HLA-DQ alleles by enzymatic amplification and allele-specific restriction fragment length polymorphism.

A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.

Base Sequence↗

[Variability of 6 Colombian strains of Trypanosoma cruzi with restriction fragment length polymorphisms (RFLP) and random amplification of polymorphic DNA (RAPD)].

Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a public health problem in Colombia. Previous reports have indicated the presence of heterogeneity among parasite populations. Six Colombian T. cruzi strains were obtained that differed by host, geographical region and transmission cycle. The genetic variability of each was compared by random amplified polymorphic DNA (RAPD), and isoenzymes. A restriction fragment length polymorphism (RFLP) was extracted using the 1.2 kb unit encoding the parasite's H2A histone as a probe. Genetic distances between the isolates varied greatly, from 0.611 to 0.99 as determined by RAPD profiles (M13F and M13R primers), between 0 and 0.81 by RFLP profiles (5 endonucleases), and between 0.10 and 0.55 by isoenzymes (13 enzymatic systems). Genetic distance matrixes derived from each of the three methods showed that Colombian strains exhibit a high degree of genetic differentiation. This may account for the broad clinical spectrum of Chagas disease in Colombia.

Animals↗

Molecular epidemiology of type 1 polioviruses isolated in Israel and defined by restriction fragment length polymorphism assay.

The genomic variability of 27 type 1 polioviruses (PV-1) isolated in Israel during 1980-1991 was examined by restriction fragment length polymorphism (RFLP) analysis of a reverse-transcribed genomic fragment amplified by polymerase chain reaction. By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles were generated for the PV-1/Mahoney and PV-1/Sabin strains and 27 wild-type isolates. The profile observed for PV-1 isolated during an outbreak in 1988 was also observed for PV-1 isolated from different places in Israel in 1982 and 1983, 1987, and 1991. This profile, characterized by the lack of the DdeI site, was different from the DdeI profile of PV-1 isolated in 1984 or in 1986 from sporadic cases of poliomyelitis. The diversity of circulating PV-1 in Israel was also confirmed by nucleotide sequence analysis. The epidemiologic information provided by the RFLP and sequence data establishes a clear epidemiologic link between epidemic and sporadic virus strains and demonstrates the power of this molecular approach to epidemiology.

Base Sequence↗

Restriction fragment length polymorphism and activation of c-Ha-ras gene in urothelial cancer.

The human c-Ha-ras gene shows restriction length polymorphism (RFLP) due to the variable tandem repetition (VTR) of DNA sequences in the 3'-flanking region. It was suggested that RFLP of the c-Ha-ras gene may be useful in detecting individuals at risk of cancer. In vitro studies showed transcriptional enhancer and promoter activities in the VTR region. In some human tumors, the loss of one c-Ha-ras allele is observed. We discuss here the possible role of VTR and allelic deletion of the c-Ha-ras allele in primary human cancers in relation to c-Ha-ras expression, and the usefulness of c-Ha-ras RFLP in the risk assessment of urothelial cancer.

Base Sequence↗