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Results for “Polymorphism, Restriction Fragment Length”

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PubMed1998

New genotypes of TT virus (TTV) and a genotyping assay based on restriction fragment length polymorphism.

A phylogenetic analysis, using the open reading frame I sequence of 93 TT viruses (TTV) obtained from various geographical areas, indicated that the virus could be classified into six different genotypes including three hitherto unreported genotypes. The high reliability of the six clusters was confirmed by bootstrap analysis. On the basis of these sequence data, a new simple genotyping assay based on a restriction fragment length polymorphism of TTV was developed. Using the enzymes NdeI and PstI, followed by cleavage with NlaIII or MseI, it was possible to distinguish between the six TTV genotypes. This system will provide the framework for future detailed epidemiological and clinical investigations.

Base Sequence↗

PubMed1988

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex.

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.

Alleles↗

PubMed2002

Identification of nontuberculous mycobacteria existing in tap water by PCR-restriction fragment length polymorphism.

This paper presents the finding of the possible cause of the high false-positive rate in acid-fast staining in histological examinations. Using acid-fast staining, culture, and PCR, acid-fast bacilli were detected in 83.7% of 49 hospital tap water samples and nontuberculous mycobacteria (NTM) were detected in 20.4% of the same 49 samples. The 10 NTM isolates were also identified to the species level using PCR-restriction fragment length polymorphism. Our findings indicate that NTM in hospital tap water are the possible cause of false positives in acid-fast staining and of nosocomial infection in immunocompromised patients.

Cross Infection↗

PubMed1994

Assignment of the zeta-crystallin gene (CRYZ) to human chromosome 1p22-p31 and identification of restriction fragment length polymorphisms.

zeta-Crystallin is a lens protein that has been associated with autosomal dominant congenital cataracts in guinea pigs and thus is a candidate for human congenital cataracts. We have assigned the zeta-crystallin gene (CRYZ) to human chromosome 1 using a Southern panel of 17 human-mouse somatic cell hybrids and regionally localized it to 1p22-p31 by fluorescence in situ hybridization. Five restriction fragment length polymorphisms were identified by analyzing the DNA from 10 unrelated, unaffected individuals. Our results will permit evaluation of its role in human cataractogenesis.

Alleles↗

PubMed2006

Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples.

At least 13 characterized Leishmania species are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment length polymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW) Leishmania species from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevant Leishmania species with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NW Leishmania species typing rapidly and easily in the field constitutes a very valuable improvement for detection of Leishmania spp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.

Animals↗

PubMed1994

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes.

The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The NotI and SfiI digestion patterns of the genomes of several V. cholerae strains belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae.

Bacterial Typing Techniques↗

PubMedDate not supplied

Molecular and genetic characterization and physical mapping of 11 new markers detecting multiallele restriction fragment length polymorphisms on the short arm of human chromosome 3.

Genetic markers with high degrees of polymorphisms are of vital importance in the construction of high resolution (2-4 cM) linkage maps of human chromosomes as specified in the short-term goals of the Human Genome Initiative. In this paper, we report on molecular and genetic characterization and physical localization of 11 new multiallele restriction fragment length polymorphism markers on human chromosome 3p. Ten of these represent three- and four-allele polymorphisms of the base substitution type probably at two adjacent restriction sites. One has been identified as a novel mini-satellite sequence comprising a variable copy number tandem repeat array of a G/T-rich 79-bp sequence. This collection of multiallele polymorphic (PIC values: 0.40-0.60) markers should prove valuable and increase the resolution power of the available chromosome 3p genetic markers.

Alleles↗

PubMed2004

Molecular differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by polymerase chain reaction-restriction fragment length polymorphism.

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Angiostrongylus↗

PubMed1987

Heterogeneity in the structural basis of the human complement C4A null allele (C4A Q0) as revealed by HindIII restriction fragment length polymorphism analysis.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes. C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes, 21-OHA and 21-OHB, and is also remarkable in the high frequency of the 'null' alleles, C4A Q0 and C4B Q0. The molecular basis for the C4A Q0 allele was studied in 26 families through restriction fragment length polymorphism (RFLP) analysis with C4 and 21-OH cDNA probes after digestion of the DNA with the endonuclease HindIII. The individuals expressing the extended haplotype HLA-A1 (of A2) Cw7 B8 C2C BfS C4AQ0B1 DR3 have a large deletion taking off the C4A and 21-OHA genes.

Alleles↗

PubMed1989

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads.

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.

Blotting, Southern↗

PubMed1991

Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: detection by exonic PCR-restriction fragment length polymorphism analysis.

von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. We report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. The maternal and paternal silent alleles are identical at two coding sequence polymorphisms as well as an intron 40 variable number tandem repeat, suggesting a possible common origin. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to approximately 70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.

Alleles↗

PubMed1990

Restriction fragment length polymorphism analysis of HLA haplotypes in families with type I diabetes mellitus.

HLA Class II polymorphisms were analysed in 27 families with at least one Type I diabetic proband using Southern blotting technique according to 10th Histocompatibility Workshop Standards. The probes used were DRB, DQA1, DQB1 and DOB. We have studied 108 haplotypes and performed segregation analysis with HLA serology and restriction fragment length polymorphism (RFLP) data and compared "affected" with "non-affected" haplotypes (not inherited by IDDM patients). RFLPs correlated well with DR and DQ serology and detected additional polymorphisms. In particular, DQB polymorphism analysis showed segregation of the DQw3 splits with 88.5% of the DR4 affected haplotypes bearing the DQw3.2 split (now DQw8) and 11.5% the DQw3.1 split (now DQw7) while in the non-affected DR4 haplotypes 33.3% were DQw3.2 and 66.6% were DQw3.1. Haplotype analysis showed that DR4-DQw3.2 was in strong linkage with the U fragment (2.1 kb Taq I) of DQA2 (DX alpha) and with the L fragment (5.4 kb BamH I) of DOB. This study confirms previous observations of DQB polymorphisms in heterozygous IDDM patients, supports the protective effect of DQw3.1 (DQw7) against the development of the disease and demonstrates the importance of DQw3.2 (DQw8) for susceptibility to Type I diabetes.

Diabetes Mellitus, Type 1↗

PubMed2000

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA.

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.

Ascomycota↗

PubMedDate not supplied

HLA-DRB and DQB typing by a combination of serology, restriction fragment length polymorphism analysis and oligonucleotide probing.

DNA sequencing has revealed extensive polymorphism within the HLA-DRB and DQB genes of the major histocompatibility complex. At least 43 alleles of the DRB loci and 13 alleles of the DQB1 locus are currently recognized. Identification of these alleles can be performed phenotypically by cellular and serological techniques or genotypically by restriction fragment length polymorphism (RFLP) analysis and sequence-specific oligonucleotide (SSO) probing of DNA amplified by the polymerase chain reaction (PCR). However, each method has its technical limitations so the tissue typing laboratory must therefore choose from the range of available techniques to maximize the efficiency and accuracy of HLA typing. In this paper we describe a scheme for the accurate determination of all the serologically defined DRB and DQB allotypes using a combination of serology, RFLP analysis and PCR-SSO probing. The efficiencies of serology versus RFLP analysis for the DR typing of 800 individuals, and RFLP analysis versus PCR-SSO probing for the DQB typing of 317 individuals, are compared and the merits of each technique discussed. This scheme, which types for both HLA-DRB and DQB with an accuracy approaching 100%, is now routinely employed in all HLA studies of disease and transplantation in our laboratory.

Adult↗

PubMed1990

Restriction fragment length polymorphism in the cytadhesin P1 gene of human clinical isolates of Mycoplasma pneumoniae.

Genomic DNA obtained from Mycoplasma pneumoniae clinical isolates spanning a 30-year period was analyzed for the presence of polymorphism in their P1 cytadhesin genes. All clinical isolates expressed a 170-kilodalton P1 protein that reacted with anti-P1 monoclonal antibodies. However, Southern blot analysis of specific M. pneumoniae isolates with subclones of the P1 structural gene revealed the presence of restriction fragment length polymorphism, permitting the classification of their P1 genes into two distinct categories.

Adhesins, Bacterial↗

PubMed1998

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism.

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn1 I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Animals↗

PubMed1997

NcoI restriction fragment length polymorphism at -308 of the tumor necrosis factor alpha (TNFA) promoter region in Korean.

Tumor necrosis factor alpha (TNFA) is a cytokine, which is secreted from activated macrophage, with a broad range of biological activities. The gene encoding TNFA is located in tandem with the TNFB gene within the HLA complex on chromosome 6p21.3. We detected a single base polymorphism in the human TNFA gene promoter region in 300 unrelated Korean individuals. The TNFA promoter region which showed a G to A transition at position of -308 was investigated by NcoI restriction fragment length polymorphism analysis. A biallelic polymorphism of TNFA gene showed fragments of 87/20 bp and 107 bp acting as TNFA*1 allele and TNFA*2 allele, respectively. The allele frequencies of TNFA*1 and TNFA*2 were 0.8783 and 0.1217, respectively. The 21.7% of heterozygosity was observed. No association between promoter region phenotypes of TNFA and the first intron phenotypes of TNFB was observed in Korean. Allele frequencies of Koreans were compared with that of Europeans.

Alleles↗

PubMed2005

Comparative typing of Campylobacter jejuni by heat-stable serotyping and PCR-based restriction fragment length polymorphism analysis.

Campylobacter jejuni has become the most common bacterial cause of human gastroenteritis worldwide. Rapid, discriminatory typing methods are required to identify potential clusters of infections. The major disadvantage of the well-evaluated and widely used Penner heat-stable serotyping method is the high level of nontypeability. The correlation of the types determined by the Penner heat-stable serotyping method and PCR-based restriction fragment length polymorphism (RFLP) analysis of the lipooligosaccharide (LOS) biosynthesis genes of C. jejuni was studied with 149 C. jejuni strains. Of these strains, 79 were patient strains belonging to 25 Penner serotypes, 60 were nontypeable patient strains, and 10 were reference strains. A 9.6-kb DNA fragment of the LOS gene cluster was amplified and digested with the restriction enzymes HhaI and DdeI. Altogether, 39 different RFLP types (including 30 HhaI profiles and 32 DdeI profiles) were identified. Type Hh1Dd1 was the most common type, with 36% of the strains and strains of 12 serotypes being of this type. A high level of discrimination was obtained, and a correlation between the Penner serotypes and the PCR-RFLP types could be seen. Also, variation in the LOS biosynthesis genes within a single Penner serotype was found. Although the PCR-RFLP method may not be sufficient to compensate for Penner serotyping, it can give valuable information about nontypeable strains and further characterize strains of common serotypes.

Bacterial Typing Techniques↗