RAPD

About: RAPD is a research topic. Over the lifetime, 15960 publications have been published within this topic receiving 360391 citations.

Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans.

Abstract: Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have emerged as effective fingerprinting methods for the infectious fungus Candida albicans. The three methods have been compared for their capacities to identify identical or highly related isolates, to cluster weakly related isolates, to discriminate between unrelated isolates, and to assess microevolution within a strain. By computing similarity coefficients between 29 isolates from three cities within the continental United States, strong concordance of the results is demonstrated for RAPD analysis, MLEE, and Southern blot hybridization with the moderately repetitive probe Ca3, and weaker concordance of the results is demonstrated for these three fingerprinting methods and Southern blot hybridization with the moderately repetitive probe CARE2. All methods were also demonstrated to be able to resolve microevolution within a strain, with the Ca3 probe exhibiting the greatest resolving power. The strong correlations demonstrated between polymorphic markers assessed by the four independent fingerprinting methods and the nonrandom association between loci demonstrated by RAPD analysis and MLEE provide evidence for strong linkage disequilibrium and a clonal population structure for C. albicans. In addition, a synapomorphic allele, Pep-3A, was found to be present in all members of one of the three clusters discriminated by RAPD analysis, MLEE, and Ca3 fingerprinting, supporting the concordance of the clustering capacities of the three methods, the robustness of the clusters, and the clonal nature of the clusters.

Evaluation of Phenotypic and Genotypic Methods for Subtyping Campylobacter jejuni Isolates from Humans, Poultry, and Cattle

Abstract: Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods.

Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars

Abstract: One hundred and two olive RAPD profiles were sampled from all around the Mediterranean Basin. Twenty four clusters of RAPD profiles were shown in the dendrogram based on the Ward’s minimum variance algorithm using chi-square distances. Factorial discriminant analyses showed that RAPD profiles were correlated with the use of the fruits and the country or region of origin of the cultivars. This suggests that cultivar selection has occurred in different genetic pools and in different areas. Mitochondrial DNA RFLP analyses were also performed. These mitotypes supported the conclusion also that multilocal olive selection has occurred. This prediction for the use of cultivars will help olive growers to choose new foreign cultivars for testing them before an eventual introduction if they are well adapted to local conditions.

Excess of non-parental bands in offspring from known primate pedigrees assayed using RAPD PCR

Abstract: The random amplified polymorphic DNA (RAPD) method allows the detection of DNA sequence polymorphisms using single primers of arbitrary sequence in the polymerase chain reaction (1). We have attempted to use this technique to assess paternity in a troop of chacma baboons {Papio cynocephalus ursinus). We have also examined individuals from known pedigrees of this species and humans. Our results demonstrate that this technique leads to an unacceptable number of non-parental bands within a pedigree, thus raising a serious concern regarding its use in paternity analysis. Twenty different RAPD oligomers obtained from Operon Ltd were used in the initial screening with the five most variable chosen for further analysis: A16, A17, A18, A19, and A20. A total of 18 field-collected baboon samples and 24 pedigree individuals, including 10 baboons and 14 humans from CEPH pedigree 1468 were examined. PCR was carried out as described by (1) with 0.4 mM MgCl2 using 5—10 ng of genomic DNA and the PCR products separated on agarose gels. Controls were run with either no Taq Polymerase or with no template DNA and produced no visible bands (Figure 1). Each RAPD primer produced a distinct pattern of amplification products consisting of 3 1 8 bands ranging in size between 0.25 kb and 6 kb in both humans and baboons (Figure 1). All primers reproducibly amplified products in some offspring which were not found in either parent (i.e., non-parental bands). We examined two nuclear families of olive baboons {Papio cynocephalus anubis) each of which included both parents and three offspring (two females, one male). The sires and dams for each family were unrelated. In the two baboon pedigrees, the average number of novel bands per parent-offspring combination was 4.4 and ranged from 1 —9 for the five primers. Similarly, a high frequency of non-parental bands was found in the CEPH pedigree for which there is no question of parentage. The average number of novel bands per parent—offspring combination in the human pedigree was 2.7 with a range of 2—4. Explanations for these results include PCR artifact and genomic mutation. Contamination of samples or reagents is unlikely given our control results and repeatability of novel bands. Polymerase slippage during replication, non-template directed addition of nucleotides by Taq polymerase and the amplification of in vitro recombinants may also generate artifactual product bands (4). Genomic mutation could also produce novel bands, but a very high rate of mutation (7—9% per band per generation) would be necessary to generate the number of non-parental fragments we observed. A mutation rate as high as 5 % per locus per gamete generation has been reported for the human minisatellite locus D1S7 (5) but most loci show mutation rates at least an order of magnitude lower. The high average band-sharing probabilities we observed for both presumably unrelated humans and baboons (62.8% and 75.9%, respectively) are not consistent with mutation being the sole source of novel bands. The RAPD technique has proven to be useful in constructing linkage maps and detecting genetic markers in a variety of organisms (1 -3 ) . Our results, however, raise serious concerns about the use of the current RAPD technique for paternity assessment. Whether due to mutation or PCR artifact, the high frequency of occurrence of non-parental bands make these genetic markers unsuitable for paternity analysis as they will lead to false exclusions. It is possible that modifications to this method may be forthcoming that will eliminate the problems we have encountered.

Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus.

Abstract: Forty-four oligonucleotide decamers were tested for their abilities to generate randomly amplified polymorphic DNA (RAPD) markers from genomic DNAs of three different isolates of Aspergillus fumigatus. Seven primers generated RAPDs that allowed the three isolates to be differentiated; one of the primers also yielded a unique RAPD pattern in each of an additional six fungal isolates, demonstrating the utility of this technique for distinguishing between A. fumigatus isolates.