Multiplex-terminal restriction fragment length polymorphism

A novel method called "multiplex-terminal restriction fragment length polymorphism (M-TRFLP)" has been recently developed which can be used for simultaneous analysis of the community composition of two or more microbial taxa (up to four). This method can also be used for microbial diagnostic purposes. For M-TRFLP analysis, primers specific to different target genes are used for multiplex-PCR, with one primer for each target being labeled with a unique fluorescent dye at its 5' end. Restriction digestion of the amplified products followed by fragment size analysis on a DNA sequencer produces profiles for targeted genes, which can be distinguished from each other by the color of the terminal fragments imparted by the unique fluorescent dye used for primer labeling. In contrast to current protocols, M-TRFLP allows multiple communities or multiple targets (genes) data to be obtained in just one reaction and therefore saves time, cost and labor. This protocol can be completed in 5-8 h.     

Renin locus restriction fragment length polymorphism

Summary The use of two genomic EcoRI fragments as probes is discussed.     

X-linked immunodeficiency with hyperimmunoglobulinemia M appears to be linked to the DXS42 restriction fragment length polymorphism locus

Summary The gene involved in X-linked immunodeficiency with hyperimmunoglobulinemia M (XHM) was localized by the use of nine restriction fragment length polymorphic (RFLP) markers covering the entire X chromosome. Multipoint linkage analysis of RFLP data obtained in a three generation XHM pedigree indicates the Xq24-q27 area around the DXS42 RFLP locus as the most likely localization of the XHM locus.     

Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.     

Choroideremia is linked to the restriction fragment length polymorphism DXYS1 at XQ13-21.

Choroideremia (McK30310), an X-linked hereditary retinal dystrophy, causes night-blindness, progressive peripheral visual field loss, and, ultimately, central blindness in affected males. The location of choroideremia on the X chromosome is unknown. We have used restriction fragment length polymorphisms from the X chromosome to determine the regional localization of choroideremia by linkage analysis in families with this disease. One such polymorphic locus, DXYS1, located on the long arm (Xq) within bands q13-q21, shows no recombination with choroideremia at lod = 5.78. Therefore, with 90% probability, choroideremia maps within 9 centiMorgans (cM) of DXYS1. Another polymorphic locus, DXS11, located within Xq24-q26, also shows no recombination with choroideremia, although at a smaller lod score of 1.54 (90% probability limit theta less than 30 cM). This linkage with DXS11, a marker that is distal to DXYS1, suggests that the locus for choroideremia is also distal to DXYS1 and lies between these two markers in the region Xq13-q24. These results provide regional mapping for the disease that may be useful for prenatal diagnosis and, perhaps ultimately, for isolating the gene locus for choroideremia.     

HLA-DR genotyping by restriction fragment length polymorphism analyses

We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.     

Evaluation of restriction fragment length polymorphism in Cucumis melo

Summary The objectives of this study were to assess the degree of restriction fragment length polymorphism (RFLP) in Cucumis melo and to determine interrelationships among cultivated varieties. Initial screening of a genomic PstI library revealed that approximately 40% of the clones were repetitive. A total of 162 unique and low-copy sequence clones were hybridized to seven diverse accesions of C. melo and a C. sativus cultivar Pacer to evaluate RFLP variation. Of these, 130 probes (80%) detected a polymorphism between C. melo accessions and C. sativus, and the majority were polymorphic with more than one enzyme digest. In contrast, only 53 probes (33%) were useful in differentiating at least one of the seven accessions. Of those, only 9% were informative with more than one enzyme digest. This indicates that within C. melo, the differences among accessions are due to infrequent base substitutions, whereas between the two species, differences are mainly due to genome rearrangements such as insertions and deletions or numerous base substitutions. Of the informative probes, 34 were used in analyzing 44 C. melo lines to establish a data base of RFLP hybridization patterns. Percent similarity based on RFLP profiles was computed among lines and analyzed by principal component analysis, to visualize relationships among lines. There were clear demarcations among, but not within, muskmelon and honeydew groups.     

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.     

EcoRI restriction fragment length polymorphism in human glycogen synthase gene

Two alleles of 10.1 and 8.1 kb of the human glycogen synthase gene have been revealed with the restriction enzyme EcoRI.     

Designation by restriction fragment length polymorphism of major histocompatibility complex class IV haplotypes in meat-type chickens

Major histocompatiblity complex (MHC) class IV haplotypes were identified in a population of meat-type chickens by restriction fragment length polymorphism (RFLP) analysis. Fourteen different haplotypes were designated on the basis of restriction patterns obtained from Southern blots of PvuII- or BglII-digested DNA, hybridized with the MHC class IV cDNA probe bg32.1. Digestion with each restriction enzyme yielded the same level of polymorphism among individuals. For each haplotype, 4–10 restriction fragments ranging from 0–8 to 8 kb were observed. Such a designation of meat-type chicken MHC class IV haplotypes enables a rapid recognition of previously defined haplotypes, is readily adjustable to additional, newly found restriction patterns and could prove useful in practical breeding programmes.     

Application of a closely linked polymorphism of restriction fragment length to counselling and prenatal testing in families with myotonic dystrophy.

The close genetic linkage between the loci for apolipoprotein CII (ApoC2) and myotonic dystrophy makes ApoC2 the closest fully validated marker for prediction of myotonic dystrophy. Application to genetic counselling and presymptomatic and prenatal prediction is reported in seven families with myotonic dystrophy, including one case in which the disorder was excluded prenatally. Only one of the families did not have members with ApoC2 genotypes that allowed prediction, but careful clinical study of older family members was found to be an important factor. ApoC2 typing of families with myotonic dystrophy should be of practical help both in prediction for asymptomatic relatives and for prenatal diagnosis in pregnancies of an affected parent.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

Residual polymerase activity-induced bias in terminal restriction fragment length polymorphism analysis

Residual activity of polymerase chain reaction DNA polymerases in restriction analyses strongly affected genetic profiling based on terminal restriction fragment length polymorphisms. Artificial fragment sizes produced as a result of 5'-overhang restriction site fill-in and addition of a terminal A may bias genetic profiling and genotyping of microbial communities. Efficient removal of polymerases retained original fragment sizes and significantly reduced this profiling bias in soil bacterial communities.     

Linkage arrangement of restriction fragment length polymorphism loci in Brassica oleracea

Summary A detailed genetic linkage map of Brassica oleracea was constructed based on the segregation of 258 restriction fragment length polymorphism loci in a broccoli × cabbage F₂ population. The genetic markers defined nine linkage groups, covering 820 recombination units. A majority of the informative genomic DNA probes hybridized to more than two restriction fragments in the F₂ population. Duplicate sequences having restriction fragment length polymorphism were generally found to be unlinked for any given probe. Many of these duplicated loci were clustered non-randomly on certain pairs of linkage groups, and conservation of the relative linkage arrangement of the loci between linkage groups was observed. While these data support previous cytological evidence for the existence of duplicated regions and the evolution of B. oleracea from a lower chromosome number progenitor, no evidence was provided for the current existence of blocks of homoeology spanning entire pairs of linkage groups. The arrangement of the analyzed duplicated loci suggests that a fairly high degree of genetic rearrangement has occurred in the evolution of B. oleracea. Several probes used in this study were useful in detecting rearrangements between the B. oleracea accessions used as parents, indicating that genetic rearrangements have occurred in the relatively recent evolution of this species.     

An additional restriction fragment length polymorphism at the thyroglobulin gene.

The study was aimed at the screening of human chromosomal DNA for restriction fragment length polymorphism (RFLP) at the human thyroglobulin (hTg) gene locus. The RFLP screening was performed in a typical way. As hybridization probes were used 5 Pst I fragments of hTg cDNA of the total length 5.1 kb pairs cloned in pBR 322. One not described polymorphism was found by using the probe hTg 10, (nucleotides from position 4830 to 5810 in the 3' flanking region of hTg). Restriction enzyme Msp I identified a single two allele polymorphism: A1: 3.5 kb and A2: 2.5 kb. Of 32 unrelated healthy individuals two were homozygous for 3.5 kb, one was homozygous 2.5 kb and 29 were heterozygous for both 3.5 kb. and 2.5 kb. Thus, the frequencies of the 3.5 and 2.5 kb Msp I alleles were 0.52 and 0.48 respectively.