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PubMed1990

Comparison of RFLP-DR beta and serological HLA-DR typing in 1500 individuals.

A group of 1522 individuals were HLA-DR-typed both by the standard serological technique and by the RFLP method. Whereas 11% (n = 164) of the serological typings were technically unsuccessful or doubtful, all typings were successful by RFLP. The results of the remaining 1358 typings revealed a serological error rate of 25%. In 16% a serological "blank" turned out to be a definable allele by RFLP, while in 9% an allele was incorrectly interpreted by serology. Of the individuals tested, 11% were HLA-DR homozygous by RFLP. Our results demonstrate an important clinical potential of RFLP typing for the typing of bone marrow transplant candidates in whom serology often fails, and for kidney transplant candidates with "blanks" or serologically "difficult" HLA antigens.

Alleles↗

PubMed2003

Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis.

Terminal restriction fragment-length polymorphism (T-RFLP) analysis was applied to characterize oral bacterial flora in saliva from 18 healthy subjects and 18 patients with periodontitis. The 16S rRNA genes (rDNAs) of oral bacteria and spirochaetes in saliva were amplified by PCR with a 6'carboxy-fluorescein (6-FAM)-labelled universal forward primer (27F) and a universal reverse primer (1492R) or the Spirochaeta-selective reverse primer. The 16S rDNAs were digested with restriction enzymes with 4 bp recognition sites (HhaI or MspI) and analysed by using an automated DNA sequencer. T-RFLP patterns were numerically analysed using a computer program. From analysis of the oral bacterial community, patterns derived from periodontally healthy subjects and patients with periodontitis were grouped into different clusters, though with some uncertainty. Samples from patients with periodontitis tended to cluster into their respective types (aggressive and chronic periodontitis), although this was not very clear. Analysis of spirochaetal community using T-RFLP showed that the patterns derived from patients with periodontitis were grouped more as compared with the analysis of the oral bacterial community. These results suggest that samples from patients with periodontitis contain an unexpected diversity. T-RFLP patterns of 16S rDNAs from saliva samples of two periodontally healthy subjects over a 5-week period showed host-specific relatively stable oral bacterial flora. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of oral bacterial flora and rapid comparison of the community structure between subjects with and without periodontitis.

Adult↗

PubMed2005

Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

Animals↗

PubMed1996

Multiple founder haplotypes of mitochondrial DNA in Amerindians revealed by RFLP and sequencing.

The mitochondrial DNA (mtDNA) of 139 individuals from eight tribes which belong to four linguistic groups of the Brazilian Amazon Region was studied both by RFLP and by sequencing of the D-loop region. RFLP analysis showed that 41 haplotypes (29%) belonged to haplogroup A, 39 (28%) to haplogroup B, 38 (27%) to haplogroup C, 19 (14%) to haplogroup D, and 2 (< 2%) could not be assigned to any of the four haplogroups. Among the 92 individuals analyzed by direct sequencing of the D-loop region, we observed 43 different haplotypes defined by 48 polymorphic points, while one haplotype could not be assigned to any of the clusters previously described. Joint analysis of data obtained by RFLP and by sequencing of mtDNA demonstrated that, regardless of the method of analysis, the mtDNA haplotypes of contemporary Amerindians cluster into four groups, similar to those previously described, even though 7% of the total sample or 12% of the haplotypes have discrepancies between results obtained by RFLP and sequencing. In addition to supporting the prevalence of four major haplogroups among contemporary Amerindians, our data are compatible with multiple founder haplotypes in each haplogroup, based on: i) a high prevalence of unusual haplotypes: ii) presence of multiple polymorphic sites shared by different haplogroups; iii) relative differences in nucleotide diversity based on RFLP or sequencing within the different haplogroups.

Base Sequence↗

PubMed2000

Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants.

The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m(2) were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as spacextime interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.

Journal Article↗

PubMed2006

Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagic Escherichia coli strains.

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.

Animals↗

PubMed2003

[Usefulness of arbitrarily primed polymerase chain reaction (AP-PCR) method for DNA fingerprinting analysis of Mycobacterium tuberculosis (MTB); comparison between DNA restriction fragment-length polymorphism (RFLP) method and AP-PCR method].

We have been analyzing cases suspected as outbreak of Mycobacterium tuberculosis in Tokyo using RFLP method. This time we analyzed 27 strains of MTB from 5 cases in two hospitals, a family, member of social activity and stuff of a corporation using both RFLP and AP-PCR methods. At 4 cases, over 80% of strains were same pattern in each cases with RFLP and AP-PCR and were identified as a patient to patients transmission of MTB. At one case, in a hospital, each strains were completely different patterns at both methods, which showed it was not a outbreak case. Results of RFLP and AP-PCR were completely same, which indicates AP-PCR is also useful and rapid method for epidemiological analysis of MTB infection as well as RFLP.

Contact Tracing↗

PubMed2005

Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays.

BACKGROUND: Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. RESULTS: A method and SPR Opt (SNP and PCR-RFLP Optimization) software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed) are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. CONCLUSION: This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As more sequence data becomes available for multiple strains and isolates of a species, automated, computational approaches such as those described here will be essential to make sense of large amounts of information, and to guide and optimize efforts in the laboratory. The software and source code for SPR Opt is publicly available and free for non-profit use at http://www.llnl.gov/IPandC/technology/software/softwaretitles/spropt.php.

Cluster Analysis↗

PubMed2006

Allelic drop-out may occur with a primer binding site polymorphism for the commonly used RFLP assay for the -1131T>C polymorphism of the Apolipoprotein AV gene.

Apolipoprotein AV (ApoAV) gene variant, -1131T>C, is associated with increased triglyceride concentrations in all ethnic groups studied. An MseI based RFLP analysis is the most commonly used method for genotyping this SNP. We genotyped a large cohort comprising 1185 Asian Indians and 173 UK Caucasians for -1131T>C using an ARMS-PCR based tetra-primer method. For quality control, we re-genotyped approximately 10% random samples from this cohort utilizing the MseI RFLP, which showed a 2.9% (3/102) genotyping error rate between the two methods. To investigate further, we sequenced the 900 bp region around the -1131T>C polymorphism in 25 Asian Indians and 15 UK Caucasians and found a number of polymorphisms including the -987C>T polymorphism. Further analysis of the -987C>T SNP showed a higher rare allele frequency of 0.23 in Asian Indians (n = 158) compared to 0.09 in the UK Caucasians (n = 157). This SNP is located 4 bp from the 3' end of the RFLP forward primer and is in weak linkage disequilibrium with -1131T>C variant (r2 = 0.084 and D' = 1). Repeated RFLP analysis of seven subjects heterozygous for -987C>T (seven times), showed discordant results with the sequence at -1131T>C SNP nearly one third (15/49) of the time. We conclude that presence of -987C>T polymorphism in the forward primer of the MseI RFLP assay may lead to allelic drop-out and generate unforeseen errors in genotyping the -1131T>C polymorphism. Our results also emphasise the need for careful quality control in all molecular genetic studies, particularly while transferring genotyping methods between various ethnic groups.

Alleles↗

PubMed1994

Survey of natural scrapie in Japan: analysis of RFLP types of the PrP gene and detection of PrPSc mainly in Suffolk sheep.

We examined the brains, spleens and/or lymph nodes of 197 mainly suffolk sheep collected from Hokkaido, and the Tohoku, Kanto and Chubu districts to detect PrPSc and thus to estimate scrapie contamination in Japan. Sixteen sheep in Hokkaido and 2 sheep in other districts that were introduced from Hokkaido were positive for PrPSc. By comparison of the frequencies of the restriction fragment length polymorphism (RFLP) types of these 18 scrapie sheep with 128 healthy sheep, we confirmed the association of specific RFLP types of the PrP gene with natural scrapie. The frequency of RFLP type I in scrapie sheep was significantly higher than that in healthy sheep and those of other types in scrapie sheep. In contrast, the frequencies of type II and VI in healthy sheep were higher than those in scrapie sheep. Therefore, type I sheep seemed to be susceptible but type II and VI sheep seemed to be resistant to natural scrapie in Suffolk sheep in Japan. Furthermore, we investigated the distribution of the RFLP types of the PrP gene in 161 sheep in Japan to learn about the genetic background of the susceptibility to scrapie. There were variations in the distribution of the RFLP types in each district.

Animals↗

PubMed1996

Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns.

The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., A. culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

Acanthamoeba↗

PubMed2000

RFLP band size standards: cell line K562 values from 1991 to 1997 proficiency studies.

Cell line K562 is the defacto forensic control material for forensic restriction fragment length polymorphism (RFLP) DNA profiling in the U.S. Fifty-one proficiency tests conducted from 1991 through 1997 enable a detailed description of RFLP measurement performance during this period. Sufficient data are available to define reference distributions for all commonly utilized and many less commonly reported genetic loci, for both HaeIII- and HinfI-based RFLP systems. The average measured size of HaeIII locus D1S7 and D5S110 bands has varied slightly over time; while relatively small, these temporal changes add to the overall interlaboratory measurement uncertainty. The characteristic standard deviation for HinfI RFLP system measurements has a nearly identical dependence on expected band size as does the standard deviation for HaeIII measurements. The ellipsoidal distance, K, is suggested for use as an RFLP data quality metric; the critical threshold value that on average excludes 1% of plausibly valid proficiency data for a given polymorphic locus is K1% = 14.2.

DNA Fingerprinting↗

PubMed2003

[Utility of RFLP analysis for tuberculosis case management in Okinawa Prefecture].

SETTING: Routine contact actions for tuberculosis (TB) case management at Public Health Centers (PHCs) in Okinawa Prefecture, Japan. OBJECTIVE: To examine the utility of RFLP analysis in routine contact actions for tuberculosis at PHCs and in the epidemiological surveillance of tuberculosis in Okinawa Prefecture, Japan. DESIGN: An RFLP analysis was conducted for all isolates of TB bacilli cultured from newly registered TB patients in Okinawa Prefecture between April 1996 and September 1997. When an identical band pattern was seen in isolates from two or more patients, the staff of the PHCs discussed the possible epidemiological link based on information collected from the patients by public health nurses at interviews in routine patient service. RESULTS: The main hospitals with TB beds, which diagnose most of the TB patients in the prefecture, and the bacteriological laboratories of the PHCs, submitted TB isolates to the Research Institute of Tuberculosis, Tokyo, for analysis. The prefecture and the PHCs conducted an additional epidemiological investigation based on the analysis results. All these activities were supervised by the Tuberculosis Surveillance Committee of the Prefecture. This organization has proven to work well, so that more than 75% of bacteriologically confirmed TB patients' strains were able be analyzed during this study period. A total of 263 samples recovered from 229 TB patients were collected and analyzed. From these, seventeen clusters have been identified, varying in size from 2 to 10. Five clusters had members that appeared to be family or friends. One cluster appeared to have a common infectious source that had not clearly been detected before the RFLP analysis. However, there was also one large cluster with members that were difficult to connect to a common source of infection, suggesting the existence of an endemic strain causing isolated infections. In many cases, links suspected through the routine contact actions by public health nurses were further supported by the RFLP analysis results. CONCLUSION: RFLP analysis can be a useful tool for TB control activities of the community level, such as in Okinawa Prefecture. First, it gives clues to understanding the epidemiological situation of TB infection in a population. Second, it facilitates detection common infectious sources that might otherwise remain undetected by routine contact actions. Third, it assist public health nurses' activities in routine epidemiological investigations of patients and their contacts in TB case management.

Cluster Analysis↗

PubMed1994

Validation studies on the forensic analysis of restriction fragment length polymorphism (RFLP) on LE agarose gels without ethidium bromide: effects of contaminants, sunlight, and the electrophoresis of varying quantities of deoxyribonucleic acid (DNA).

This study was designed to analyze the effects of sunlight, various contaminants (those found typically in forensic samples) and the electrophoresis of varying quantities of DNA on the restriction fragment length polymorphism (RFLP) patterns produced from DNA isolated from blood and semen stains. The DNA RFLP patterns were obtained following Hae III restriction enzyme digestion, low electroendosmotic (LE) agarose gel electrophoresis (in the absence of ethidium bromide), Southern transfer, hybridization with DNA probes detecting highly polymorphic variable number of tandem repeats (VNTRs) and autoradiography. Computer assisted image analyses were used to detect variations in RFLP band sizes in relation to control samples. Comparisons between the samples were made for the presence of high molecular weight DNA, the ability to achieve a complete restriction digestion, and the RFLP fragment sizes obtained. The results demonstrate that high molecular weight DNA can be obtained when blood and semen stains are subjected to environmental and contaminating factors. The RFLP allele sizes were not significantly affected by environmental conditions, contamination factors or by loading varying amounts of DNA. This study serves to further document the reliability and validity of DNA typing for forensic applications.

Alleles↗

PubMed1995

Comprehensive typing of HLA-DPB1 alleles by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Comprehensive typing of 53 HLA-DPB1 alleles was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method using 78 polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) defined DNA specimens (14 retrospective, 64 prospective). A single primer pair was used to amplify the second exon to obtain DPB1-specific amplified product of 294 bp. A combination of RFLPs and cleaved/uncleaved patterns of various endonucleases was employed to resolve DPB1 alleles. A panel of 13 endonucleases (RsaI, Sau96I, BsrBI, DdeI, BsaJI, BssHII, ScaI, ScaI, BbvI, BsgI, FokI, Bsp1286I and BstUI) yielded unique RFLP patterns for all but 2 pairs of DPB1 alleles. However, these remaining 2 pairs of rare alleles could be resolved by an additional digestion with AciI (DPB1*3901 from 4001 and DPB1*4901 from 5301). The unique RFLP patterns of 21 DPB1 alleles using PCR-SSOP typed DNA specimens had been verified. Of the 1,378 possible heterozygotic patterns, 69 pairs and a triplet had been identified that would yield identical RFLP patterns. However, all but one pair, DPB1*3901/5301 from 4001/4901, of these heterozygotes could be resolved by double digestions with appropriately selected endonucleases from the panel used here. Thus, PCR-RFLP remains a simple and effective method for high resolution DPB1 typing.

Alleles↗

PubMed1996

Restriction fragment length polymorphism (RFLP) analysis on DNA from human compact bone.

DNA typing techniques primarily identify specific genetic markers that are highly polymorphic within a population and have found great utility in forensic science. The established DNA identification protocol, termed restriction fragment length polymorphism (RFLP), has been admitted as physical evidence in the investigation of crimes such as assault, sexual assault, and homicide. The limitation associated with this procedure concerns the integrity of the genetic material. This study sought to evaluate human bone as a source material for DNA identification following exposure to common forensic field conditions. Often, with the onset of decomposition and eventual disarticulation of a body, soft tissues, hair and teeth may not be recovered. The significance of this study lies in the fact that, within forensic anthropology, human bone represents the most biologically stable evidence and is sometimes all that remains after periods of exposure. Genomic DNA was extracted from human bone following exposure to surface deposit, shallow burial, and fresh water immersion. Samples were collected over a three month time course and analyzed by spectrophotometry and agarose gel electrophoresis as well as RFLP analysis. The data suggest that high molecular weight DNA may indeed be extracted from human bone and typed by RFLP analysis for use in forensic identification. Under simulated forensic field conditions, the severity of DNA degradation was in the order of fresh water immersion > shallow burial > surface deposit. Genomic DNA from bone deposited on the desert surface for up to 4 weeks was detected by RFLP analysis. No spurious bands were detected in any specimens, and to the extent that bands were still present, the RFLP patterns matched. These findings demonstrate that human bone can be a reliable source of genomic DNA, and that bone recovered from surface deposit is the most desirable for use in forensic identification.

Bone and Bones↗

PubMed1992

Correlation of L-myc RFLP with metastasis, prognosis and multiple cancer in lung-cancer patients.

For further study of the correlation of L-myc restriction-fragment-length polymorphism (RFLP) and metastasis of lung cancer to lymph nodes or other organs at the time of surgery, L-myc RFLP was analyzed in 252 Japanese lung-cancer patients. A close correlation between L-myc RFLP and metastasis was confirmed in this large number of patients (p = 0.01). The correlation was particularly pronounced in cases of adenocarcinoma and squamous-cell carcinoma. Poor prognosis (additional metastases after surgery) was observed in lung-cancer patients with L-S (identified as long and short bands produced with EcoRI) and S-S type L-myc RFLP. In addition, the death rate of lung-cancer patients with the L-S and S-S types was greater than that of those with the L-L type. Lung-cancer patients of the L-S and S-S types had almost 4 times higher incidence of multiple cancer in the lung, pharynx and other organs than those with the L-L type. Our results indicate that, in patients with lung cancer, genetic disposition with respect to the L-myc gene influences the extent of metastasis, the incidence of multiple cancers and prognosis.

Combined Modality Therapy↗

PubMed1991

Usefulness of chromosome 19 RFLP haplotypes in the diagnosis of myotonic dystrophy.

Three DNA probes (APOC2, PSC11, and LDR152) detecting RFLP polymorphisms were used to test the usefulness of the RFLP approach in myotonic dystrophy (MD) families from the isolated Finnish population. The informativeness of these polymorphisms did not differ from that reported in more mixed populations: in the 13 families of the study most of the 79 meiotic events studied were informative. One known recombinant is included in the study. The highest lod score obtained in the multilocus linkage analysis was z = 5.941 at recombination fraction theta = 0.02. The RFLP results significantly facilitated genetic counseling in problematic cases among the families studied. Although evidence could be found for linkage disequilibrium of the RFLP haplotypes formed in Finnish MD patients, our results do not exclude the possible existence of more than one ancient MD mutation in this population.

Chromosomes, Human, Pair 19↗