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PubMed1999

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.

Animals↗
PubMed1992

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.

Alleles↗
PubMed1992

Molecular epidemiology of group B streptococcal infections: use of restriction endonuclease analysis of chromosomal DNA and DNA restriction fragment length polymorphisms of ribosomal RNA genes (ribotyping).

Epidemiologic investigation of group B streptococcal (GBS) infections has been limited by the lack of a discriminatory typing system. Therefore, the use of restriction endonuclease analysis of chromosomal DNA (REAC) and DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) to subtype molecularly GBS isolates associated with human invasive disease was investigated. Chromosomal DNA of selected GBS isolates was initially digested with 24 different restriction enzymes. HhaI gave the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Ribotyping and REAC differentiated among isolates of the same and different serotypes. Nine ribotype patterns were noted among the 76 isolates studied, including 4 among serotype Ia/c and 4 additional ribotypes among serotype III isolates. Epidemiologically related isolates (e.g., mother-infant or twin-twin pairs) had identical REAC and ribotype patterns. Epidemiologically unrelated isolates with the same ribotype usually had different REAC patterns, suggesting that REAC may be a more sensitive technique for strain differentiation. REAC and ribotyping were reproducible and proved to be successful molecular epidemiologic methods for subtyping GBS.

Adult↗
PubMed1990

Cloning of a DNA fragment displaying restriction fragment length polymorphisms between the genomes of spontaneously hypertensive and Wistar-Kyoto rats.

A 3 kilobase (kb) EcoRI fragment cloned from the genome of the spontaneously hypertensive rat (SHR) displayed restriction fragment length polymorphism (RFLP) compared with the genome of the Wistar-Kyoto rat (WKY) when total genomic Southern blot analysis was performed for two restriction enzymes, PstI and PvuII. Sequencing of the DNA fragment cloned from genomic SHR and WKY libraries revealed that this 3 kb EcoRI fragment harbours three point mutations. Two of them (C to T and A to T) are situated in the middle of the restriction sites for PstI and PvuII, thus disrupting the recognition sites for these enzymes in the SHR genome. Southern blot analysis using total complementary (c) DNA obtained from cDNA libraries of aortic smooth muscle cells from SHR and a whole WKY kidney, with this 3 kb EcoRI fragment as a probe, showed polymorphic bands suggesting that these point mutations are reflected in the sequences of messenger (m) RNA transcribed from the gene encoded in this 3 kb fragment. Detection of two bands by a Northern blot analysis for RNA from various SHR tissues indicates that this 3 kb fragment is actively transcribed in vivo.

Animals↗
PubMed1999

[Analysis of rRNA gene restriction fragments length polymorphism of Leptospira in China].

Sixty-four Leptospira international and domestic reference strains, which belonging to fifty-four serovars, and twenty-seven field strains were examined by using EcoR I restriction endonuclease analysis of genomic DNA and restriction fragments length polymorphism of rRNA gene, fifty-six Leptospiral ribotypes(RTs) were described. Most serovars gave specific patterns. Serovars in the same serogroup possess common core-segments, but we found RTs of reference strains from China and other countries are different. Most field strains have RTs with correspond of reference strains, only a few bands were shown different if RTs were different. A notable result was that the field strains of serovar pomona have the same Rt as the international reference strain but different from the domestic reference strain.

China↗
PubMed1992

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.

Animals↗
PubMed1989

Definition of DRw10 by restriction fragment length polymorphism.

To better define the presence of the DRw10 haplotype which has sometimes proved difficult to type by using serologic reagents, Southern blot analysis was performed on seven DRw10 heterozygous individuals with rheumatoid arthritis. Using the restriction enzymes Taq I or BamH I, the restriction fragment length polymorphism (RFLP) pattern for the DRw10 haplotype was clearly distinguishable from that of other DR alleles. Digestion with Taq I revealed a unique DR beta/Taq I 12.20 fragment. A characteristic DR beta/Taq I 4.60 fragment was also present only in DRw10 and DR1 haplotypes. Digestion with the restriction enzyme BamH I revealed a DR beta/BamH I 5.07 fragment also present in DRw10 and DR1 haplotypes, and a DR beta/BamH I 4.30 fragment shared with the DRw52 and DR2 haplotypes but not found in DR1 haplotypes. The pattern was readily distinguished from those given by the haplotypes DR4, 7 and w9. Family studies of five individuals demonstrated appropriate segregation of the restriction fragments. In particular, segregation of DRw10 haplotypes from DR1 haplotypes was clearly shown in a family in which the DRw10 haplotype was associated with rheumatoid arthritis in two individuals. Southern blot analysis proved to be a useful alternative method for identifying the DRw10 allele in certain combinations where this allele has been difficult to define serologically.

Arthritis, Rheumatoid↗
PubMed1989

HLA-D typing of heterozygotes using restriction fragment length polymorphism in the HLA-DQ gene region on the basis of standard band patterns derived from HLA homozygotes.

Genomic DNAs from 37 Epstein-Barr virus-transformed HLA-D homozygous cell lines with different Dw specificities, Dw1-Dw23, and DKT2 were digested with four different restriction endonucleases (EcoRI, PstI, TaqI, and MspI) and hybridized to DQ alpha and DQ beta cDNA probes. Polymorphic patterns of multiple fragments correlating with Dw, DQ, and DR haplotypes were detected, and the restriction fragment length polymorphism standard pattern specific for each Dw, DQ, and DR specificity could be defined. The polymorphic fragment patterns of HLA-D heterozygotes were predicted simply by the summation of two standard patterns of HLA-D homozygotes and utilized to identify HLA-D specificities of 25 normal individuals, who are HLA heterozygotes in most cases. The HLA-D, -DR, and -DQ specificities defined by this DNA typing were compared with those assigned by serologic and cellular typing. There was good correlation, allowing the application of accurate genotyping by DQ alpha and DQ beta cDNA probes to HLA class II typing of HLA heterozygotes.

Base Sequence↗
PubMed1999

Development of a ssRNA internal control reagent for an infectious bursal disease virus reverse transcription/polymerase chain reaction-restriction fragment length polymorphism diagnostic assay.

Infectious bursal disease virus (IBDV), family Birnaviradae, is the etiologic agent of a commercially important, globally distributed, contagious, immunosuppressive disease of young chickens. A restriction enzyme-compatible ssRNA internal control was developed for an IBDV reverse transcription/polymerase chain reaction-restriction fragment length polymorphism (RT/PCR-RFLP) diagnostic assay. An 841-bp bacteriophage-lambda DNA fragment was directionally ligated to 3' and 5' oligonucleotide linkers containing the IBDV RT/PCR target primer sequences. A pGEM-3Zf (+) transcription vector containing the internal control construct was used in an in vitro transcription reaction to produce ssRNA. After RT and PCR amplification, the transcripts produced an 882-bp cDNA product, larger than, co-amplifiable with, and free of the restriction sites used to prepare RFLP patterns of the 743-bp IBDV cDNA target product. The limit of detection of the transcripts in the RT/PCR test is 3.2 femtograms. With the internal control, a test inhibition rate of 7.7% (20/261) was determined for the IBDV RT/PCR assay. By identifying inhibited tests, the assay was improved through a reduction in the number of false-negative results.

Animals↗
PubMed2001

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.

Aspergillosis↗
PubMed1989

DQA1 restriction fragment length polymorphisms and insulin-dependent diabetes mellitus: a BglII fragment labels a subset of B8,DR3 haplotypes uniquely associated with insulin-dependent diabetes mellitus.

Class II restriction fragment length polymorphism studies of 38 pedigrees with multiple cases of insulin-dependent diabetes mellitus revealed the existence of a DQA1-related polymorphism that distinguishes two kinds of HLA-B8,DR3 haplotypes. One of these, characterized by the presence of DQA1-BglII 7.20 kb, was present in all 14 examples inherited by patients and in 6 of the 12 B8,DR3 haplotypes not so inherited. The apparently complete association between the presence of this fragment and the "affected" status of B8,DR3 haplotypes (p = 0.004) was confirmed in a separate group of 26 simplex pedigrees selected for the presence of this haplotype in the respective probands (combined p less than 0.0001).

Diabetes Mellitus, Type 1↗
PubMed1988

Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha- and beta-chain cDNA probes.

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.

Animals↗
PubMed1990

Growth hormone restriction fragment length polymorphisms that segregate with 42-day live weight of mice.

The known correlation between growth hormone levels and growth rate in a number of species prompted us to examine if polymorphic restriction fragment alleles at the growth hormone locus in mice might be associated with differentiable rates of growth. An F2 population of mice was generated from crosses between a line selected for high 42-day weight and an unselected control line. The original selected and control lines exhibited mean 42-day weights of 30.6 +/- 3.8 and 20.5 +/- 2.6 g, respectively. Since the two lines also differed with respect to the restriction fragments detected by hybridization to a rat growth hormone cDNA probe, an analysis of the F2 generation was carried out to determine whether this polymorphism could be considered a quantitative trait locus for 42-day weight. The results of the analysis indicated that a polymorphic HindIII restriction fragment was correlated (P less than 0.05) with 42-day weight. However, the allele that was positively correlated with weight was the one that was fixed in the original control line, rather than the one from the selected line. While these findings support the potential use of restriction fragment length polymorphisms in quantitative trait evaluation of livestock, they also emphasize the requirement for testing such potential quantitative trait loci in the appropriate genetic background.

Animals↗
PubMedDate not supplied

Detection of Mycoplasma gallisepticum, M. synoviae, and M. iowae by multi-species polymerase chain reaction and restriction fragment length polymorphism.

A single set of oligonucleotide primers was designed from known 16S ribosomal RNA (rRNA) sequences of Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. iowae (MI). This set of primers selectively amplifies a 780-base-pair DNA fragment within the 16S rRNA gene of MG, MS, and MI but does not amplify other avian mycoplasmas or other bacteria. The detection limit of the multi-species polymerase chain reaction (PCR) was approximately 100 mycoplasma (MG, MS, MI) colony-forming units per PCR reaction. The PCR product was differentiated by restriction fragment length polymorphism with the restriction enzymes HpaI, HpaII, and MboI. Preliminary results from field samples suggest that this technique could be a useful and rapid diagnostic test for the detection of these three pathogenic poultry mycoplasmas.

Animals↗
PubMed1989

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines.

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.

Animals↗
PubMed1996

Plasmid profiles, restriction fragment length polymorphisms and O-serotypes among Vibrio anguillarum isolates.

A total of 279 Vibrio anguillarum strains were serotyped and examined for plasmid content. Plasmids were subjected to digestion with restriction enzymes. Most strains belonged to serogroup O1 (39%) and O2 (16%). In total 164 strains (53%) carried plasmids. Of the O1 and O2 isolates, 92% and 30%, respectively, carried one or more plasmids. Restriction fragment length polymorphism (RFLP) analysis of plasmid DNA indicated that plasmids belonged to several groups. Each group seemed to be restricted to a single O-serovar. The largest group was the pJM1-like plasmids among most serovar O1 strains. Most of these plasmids were about 67 kb like the pJM1 plasmid, but various derivatives ranged from 26-77 kb. RFLP studies of the 67 kb plasmids revealed 17 different restriction patterns. Some patterns were dominant among European strains whereas others were dominant among North American strains. The results confirmed the applicability of O-serotyping together with plasmid profile and restriction analysis of plasmids for typing of V. anguillarum. They also indicated that plasmids among strains which belonged to the traditional fish pathogenic serogroups, O1 and O2, showed more homology than did strains from most other serogroups, that were usually non-pathogenic, environmental bacteria.

Plasmids↗
PubMed2001

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA.

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.

Animal Feed↗