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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↗
PubMed1993

Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms.

Chromosomal DNA from 37 Leptospiracae representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, ClaI and EcoRI. The Southern blots were hybridized with a biotinylated E. coli 1.5 kb 16S rDNA probe and gave 36 reproducible and unique patterns. With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no. 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, ClaI and EcoRI restriction fragment length polymorphisms (RFLP). L. interrogans and L. borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L. parva incertae sedis were distinct and separate. Strains representing L. biflexa sensu lato presented divergent RFLP patterns. A porcine isolate was identified to be L. interrogans pomona Pomona.

Base Sequence↗
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↗
PubMed1997

Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.

DNA, Bacterial↗
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↗
PubMed1993

Restriction fragment length polymorphisms of medicinal plants and crude drugs. II. Analysis of Glehnia littoralis of different geographical origin.

Total DNA was extracted from leaves of Glehnia littoralis belonging to various geographical strains with different furanocoumarin composition, and digested with restriction enzymes. Hybridization of digoxigenin-labeled probe containing rice ribosomal DNA to the digested DNA showed no difference in the restriction fragment length polymorphism (RFLP) profiles among the plants of different geographical origin. It is concluded that genetic diversity among the geographical strains of G. littoralis is so narrow as to be incapable of detecting RFLPs using rDNA as a probe.

DNA↗
PubMed2001

Species identification in seven small millet species using polymerase chain reaction-restriction fragment length polymorphism of trnS-psbC gene region.

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR-RFLP). PCR-RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR-RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.

DNA, Chloroplast↗
PubMed1990

Restriction fragment length polymorphisms in the ribosomal gene spacers of Trypanosoma cruzi and Trypanosoma conorhini.

The ribosomal RNA genes of two species of Trypanosoma, Trypanosoma cruzi, the etiological agent of Chagas' disease, and Trypanosoma conorhini, a non-pathogenic rodent trypanosome, were cloned and partially characterized. The physical maps derived for their rRNA genes were similar throughout the region that encompasses the SSU-and LSU-rRNA coding sequences. However, the non-transcribed spacer DNA of both T. cruzi and T. conorhini was found to be polymorphic for several restriction enzyme sites. We show that strains of T. cruzi can be typed according to the characteristic restriction fragment length polymorphism of their NTS DNAs.

Animals↗
PubMed1998

Typing of human Mycobacterium avium isolates in Italy by IS1245-based restriction fragment length polymorphism analysis.

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.

Acquired Immunodeficiency Syndrome↗