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Results for “Polymorphism, Restriction Fragment Length”

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PubMed2001

Mycobacterium bovis: polymerase chain reaction identification in bovine lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism.

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Animals↗

PubMed1989

Restriction fragment length polymorphism analysis of hematopoietic cells following successful treatment of relapsed acute lymphoblastic leukemia following bone marrow transplantation.

Despite aggressive therapy for leukemia in the form of bone marrow transplantation (BMT) relapse occurs in a significant number of cases. The origin of the leukemic relapse, whether it is of donor or recipient origin, and how best to treat the patients continue to pose problems for the clinician. In this paper we present a case in which the cytogenetics suggested that the relapse was of donor origin; however, molecular analysis revealed that the leukemic population was of host origin. The leukemic relapse following the BMT was treated with a second BMT. This resulted in a remission of 28 months after which leukemic relapse was again diagnosed. Using conventional chemotherapy it was possible to obtain another complete remission. This case illustrates a pitfall to cytogenetic analysis and two contrasting methods of dealing with leukemic relapse following BMT.

Adult↗

PubMed1993

[The association of HincII/low density lipoprotein receptor (LDLR) restriction fragment length polymorphism (RFLP) with diabetes mellitus and its lipid phenotype with PCR gene amplification].

The association of low density lipoprotein receptor (LDLR) gene HincII RFLP with diabetes mellitus and its lipid metabolism was studied in 196 Chinese with PCR gene amplification. 16 IDDM and 75 NIDDM were included. The most common genotype and allele frequencies of NIDDM were H2H2 (0.78) and H2 (0.89) respectively, and no significant differences were found in comparison with the normal control. The NIDDM with low LDL level (< 1.3 mmol/L) had less H2H2 type and H2 frequencies. Allele 1 (H1) was possibly related to the higher level of serum cholesterol, but allele 2 (H2) was quite the reverse. The phenotype of lipid metabolism was partially determined by the genotype. LDL, tc and tg level of NIDDM were significantly higher than the normal control (P < 0.001, 0.001, 0.05 respectively), indicating that NIDDM was accompanied by disturbance of lipid metabolism.

Adolescent↗

PubMed1987

Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family.

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.

DNA↗

PubMed1996

A novel Tth111I restriction fragment length polymorphism (RFLP) allows tracing of X-chromosome inactivation in the (Xid) heterozygote.

The X-linked immunodeficiency (Xid) in CBA/N mice serves as a model for the X-linked agammaglobulinemia (XLA) syndrome in man. X-chromosome inactivation in F1 heterozygotes derived from CBA/N (Xxid/Xxid) and B6.Pgk-1a (X+/Y) was investigated by monitoring the methylation status of the individual Pgk-1 alleles, Pgk-1b and Pgk-1a, respectively, using a novel Tth111I RFLP. Results indicate that in circulating B lymphocytes of female heterozygotes, only the X chromosomes carrying the normal alleles (X+) are active (nonrandom inactivation of the X chromosome), whereas in non-B cells both the X chromosomes (X+ and Xxid) are active (random inactivation of the X chromosome). These results were further confirmed by direct evaluation of transcription of the Btk gene, the gene mutated both in Xid and in XLA.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1991

Restriction fragment length polymorphisms associated with the factor VIII and factor IX genes in Polynesians.

New Zealand Maoris (72 X chromosomes) have been compared with Pacific Island Polynesians (121 X chromosomes) and Caucasian New Zealanders (51 X chromosomes) as a control group to determine the allelic frequency of six RFLPs associated with the genes for two X linked diseases (haemophilia A and haemophilia B). RFLPs examined were BclI, XbaI, and BglI within the factor VIII gene, the factor VIII extragenic TaqI system, and the factor IX intragenic TaqI and XmnI sites. The information obtained facilitates the design of strategies for both carrier detection and prenatal diagnosis of haemophilia A within these groups. Strong linkage disequilibrium was observed between the factor VIII BclI and XbaI sites in Polynesians. Genetic counselling for Polynesians with haemophilia B continues, however, to rely on phenotypic diagnosis. The RFLP data from the two separate loci on the X chromosome in Polynesians show similarities with Chinese and Japanese populations, reinforcing theories of an early Polynesian ancestry originating in east Asia.

Alleles↗

PubMed1998

Simultaneous polymerase chain reaction restriction fragment length polymorphism identification of the factor V Leiden allele and the prothrombin 20210A mutation.

A novel mutation in the 3' untranslated region of the prothrombin gene, prothrombin 20210A, recently has been identified. This mutation is associated with increased serum prothrombin levels and an increased risk for venous thromboembolism. Patients who carry a mutation in the factor V gene (factor V Leiden) have also been demonstrated to be at increased risk for venous thromboembolism, and previous studies have identified a population prevalence of approximately 5% to 10% for the factor V Leiden allele. To simply and reliably identify patients who carry both genetic defects, a novel assay was developed that simultaneously determines the genotype of patients for the factor V Leiden allele and the prothrombin 20210A mutation. Representative samples (samples positive and negative for each mutation and a "double mutant") were then subjected to this single-tube genotyping assay. The results indicate that the simultaneous genotyping of these mutations will readily characterize the allelic status of patients for the two most frequent genetic mutations in the coagulation cascade.

3' Untranslated Regions↗

PubMed1987

Characterization of DR blank alleles by restriction fragment length polymorphism (RFLP).

Using a combination of conventional DR serology and RFLP analysis of DR beta and DQ beta, we have been able to identify two different types of DR antigens which belong so far to the DR blank group. The antigen DR-LOT is found on a haplotype A29, Bw60, Cw3, DRblank, DRw52, DQw1. The DR beta-EcoRI RFLP pattern of this haplotype is different from the patterns observed for DR1, DR2, DR3, DR4, DR5, DRw6, DR7, DRw8, DRw9, DRw10, and appears to be composed of a combination of DR2 and DRw6. The DQ beta-EcoRI pattern shows that this haplotype carries the DQw1 split DQR2.6. The second DR blank antigen which we found in a total of five individuals (three unrelated persons and two parents) on B35 positive haplotypes is characterized by a DR beta-EcoRI RFLP pattern indistinguishable from DR1 and by negative reaction with anti-DR1 sera. This antigen appears to be identical to what has been described by Cambon-Thomsen et al. (1986) and Bidwell et al. (1985) as HLA-DR-BON and DR"BR" respectively. We have demonstrated that this antigen is in strong linkage disequilibrium with the DQw1 split DQR1.

Alleles↗

PubMed1998

Restriction fragment length polymorphism and divergence in the genomic regions of high and low recombination in self-fertilizing and cross-fertilizing aegilops species.

RFLP was investigated at 52 single-copy gene loci among six species of Aegilops, including both cross-fertilizing and self-fertilizing species. Average gene diversity (H) was found to correlate with the level of outcrossing. No relationship was found between H and the phylogenetic status of a species. In all six species, the level of RFLP at a locus was a function of the position of the locus on the chromosome and the recombination rate in the neighborhood of the locus. Loci in the proximal chromosome regions, which show greatly reduced recombination rates relative to the distal regions, were significantly less variable than loci in the distal chromosome regions in all six species. Variation in recombination rates was also reflected in the haplotype divergence between closely related species; loci in the chromosome regions with low recombination rates were found to be diverged less than those in the chromosome regions with high recombination rates. This relationship was not found among the more distantly related species.

DNA, Plant↗

PubMed1989

Carrier detection and early diagnosis of Wilson's disease by restriction fragment length polymorphism analysis.

Wilson's disease, a rare autosomal recessive disorder, has been recently mapped to the long arm of chromosome 13 (q14.1). In this study, we carried out linkage analysis between three chromosome 13 DNA markers, D13S1, D13S10, D13S2, the locus for the red cell enzyme esterase D (ESD), and the Wilson's disease locus (WND) in 17 Wilson's disease families of Italian descent, mostly from Sardinia. We confirmed a tight linkage [theta = 0.00, Z (theta) = 4.07] between the WND and ESD loci, and provided suggestive evidence for linkage [theta = 0.00, Z(theta) = 1.85] of the WND locus with D13S10. Multipoint linkage analysis indicated the following order: centromere-D13S1-D13S10-WND-ESD-D13S2. RFLP analysis at these two loci in our families allowed us either to define the carrier status (50%) or to exclude the homozygous state (25%) in the great majority of unaffected sibs.

Chromosomes, Human, Pair 13↗

PubMed1989

Interstrain restriction fragment length polymorphism in the c-src correlates with Na,K cotransport and calcium content in hybrid rat erythrocytes.

Twenty-six F2 hybrids between spontaneously hypertensive (SHR) and normotensive control (WKY) rats [(SHR x WKY)F2] were segregated according to their c-src genotype into SS and WW homozygous groups, corresponding to SHR or WKY and a WS heterozygous group. The Na,K cotransport in erythrocytes in the WW group was equal to that of WKY and differed significantly from that of the WS and SS groups (the rate of Na,K cotransport in the latter groups was close to that of the SHR). The calcium content of the erythrocytes in the WW group was equal to that of the WKY, but lower than that of the WS and SS groups which, in turn, was significantly lower than that in the SHR, indicating polygenic control of the trait. We concluded that the c-src locus itself or some other loci inherited in conjunction with the c-src determines the increase in Na,K cotransport and in calcium content in erythrocytes of SHR.

Animals↗

PubMed1998

[Mismatched PCR combined with restriction fragment length polymorphism assay for identification of hot spot for mutation in core promoter sequence of hepatitis B virus in Chinese isolates].

OBJECTIVES: To investigate the prevalence of the core promoter variant in Chinese HBV carriers and to explore the clinical and epidemiological problems of the variant. METHODS: A novel method for detecting the double mutation in core promoter was developed. By using an antisense primer that starts at nt 1765 and bears a mismatch at nt 1767, we introduced an artificially created Bcl I cleavage site if the double mutation was present. The prevalence and clinical significance of mutation in the core promoter were investigated in 114 Chinese individuals infected with HBV. RESULTS: Serum samples from 37 asymptomatic carriers and patients harbored core promoter mutant, some with precore mutation. The prevalence was higher in carriers and patients without HBeAg than those with HBeAg. The results in the PCR-RFLP assay perfectly agreed with the data from direct sequencing. CONCLUSION: The presumed core promoter mutations resulting in decreased HBeAg synthesis might be one of the factors leading to persistence of HBV infection and active liver disease.

Adult↗

PubMed1997

A restriction fragment length polymorphism assay that differentiates human N-acetyltransferase-1 (NAT1) alleles.

Currently there is much interest in the N-acetyltransferase-1 (NAT1) genetic polymorphism and its relationship to cancer. Previous studies have described methods to distinguish NAT1 alleles through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) and/or allele-specific amplification. However, these methods detect at most only four of the NAT1 alleles identified in human populations. In this paper we describe a PCR-RFLP-based assay that differentiates among eight human NAT1 alleles (NAT1*3, *4, *5, *10, *11, *14, *15, *16). This method should prove useful in molecular epidemiological studies investigating associations between NAT1 genotype and cancer.

Alleles↗

PubMed1998

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.

Anaplasmataceae↗

PubMed2001

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C190 and 100 alleles.

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.

Alleles↗

PubMed1994

Restrictive fragment length polymorphisms of markers 3'HVR and 24-1 and their applications in gene diagnosis of adult polycystic disease in Chinese.

Adult polycystic kidney disease was diagnosed with pedigree analysis using polymorphic DNA markers linked to the disease gene in 5 families. The markers were thoroughly characterized in the population. We noted two important markers, 3'HVR and 24-1, which flank the disease gene PKD1 in normal Chinese. Southern hybridization was used, and numerous 3'HVR/PvuII polymorphic allelic bands varied from 1.4 kb to 8.0 kb with a positive skew distribution. They were divided into 11 groups with an interval of 0.6 kb and a heterozygosity rate of 0.86. Allele frequencies were observed. When the probe 24-1 was hybridized with genomic DNA digested by TaqI, two alleles, B1 and B2, were detected with frequencies of 0.76 and 0.24, respectively. B1 was 3.8 kb in size and B2 was composed of 2 bands, 1.5 kb and 1.3 kb. The polymorphism information content was found to be 0.297. Using these two markers for linkage analyses of 5 affected families, we confirmed the linkage between 3'HVR and the disease gene PKD1 and made presymptomatic diagnosis of the members at risk.

Alleles↗

PubMed1989

Restriction fragment length polymorphism of cardiac myosin heavy chain gene in rats and its strain distribution.

The distribution of an RFLP in EcoRI fragments of the cardiac myosin heavy chain gene among 29 strains of laboratory rats was examined. Southern blot hybridization of rat genomic DNAs with rat cardiac myosin heavy chain cDNA as a probe demonstrated an interstrain variation in one of eight EcoRI fragments. Of the 28 inbred strains examined, 10 had a fragment of 10 kbp, whereas 18 had a fragment of 7.5 kbp. The 15 samples of the remaining strain (Iar: WI outbred stock) had fragments of either 7.5 kbp or 10 and 7.5 kbp, indicating that this strain has maintained heterogeneity of these fragments.

Animals↗

PubMed2006

TRFMA: a web-based tool for terminal restriction fragment length polymorphism analysis based on molecular weight.

UNLABELLED: TRFMA provides a Web environment for analyzing T-RFLP results based on molecular weights of the fragments, rather than the numbers of nucleotides, to increase accuracy. The 16S rRNA data are saved as an XML file containing around 650 sequences (light version) and a MySQL database containing around 50 000 sequences (full version), which are connected to Web server via PHP5 and manipulated on an Internet browser. AVAILABILITY: TRFMA is freely available at http://myamagu.dent.kyushu-u.ac.jp/bioinformatics/trfma/index.html and can be downloaded from the same site.

Algorithms↗