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Quantitative PCR technique for the identification of microrearrangements of the AZFc region.

ABSTRACT: PURPOSE: The AZFc region spans about 3.5 Mb and contains many amplicons causing recombination events. Several papers have reported the occurrence of AZFc partial deletions resulting from non allelic homologous recombination (NAHR) ("gr-gr", "b1-b3" or "b2-b3" deletions), particularly in infertile patients. DAZ genes are present in 4 copies and rearrangements involve a modification of the number of DAZ genes. METHODS: In addition to STS plus/minus PCR, we developed a quantitative technique using real time PCR (Q-PCR) to determine the number of DAZ genes. Fourteen DNA controls were selected to validate the use of Q-PCR to detect AZFc microrearrangements, and sperm DNA samples from 30 fertile men were studied. RESULTS: Rearrangements of 14 controls were well identified with Q-PCR, and 2 AZFc partial deletions were detected in fertile men (1 "gr-gr" and 1 "b2-b3"). CONCLUSION: Q-PCR represents a well-adapted method to detect microrearrangements of the Y-chromosome, complementary to STS analysis.

SUBMITTER: Roze V

PROVIDER: S-EPMC3454972 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Quantitative PCR technique for the identification of microrearrangements of the AZFc region.

Rozé Virginie V Bresson Jean Luc JL Fellmann Florence F

Journal of assisted reproduction and genetics 20070405 6

<h4>Purpose</h4>The AZFc region spans about 3.5 Mb and contains many amplicons causing recombination events. Several papers have reported the occurrence of AZFc partial deletions resulting from non allelic homologous recombination (NAHR) ("gr-gr", "b1-b3" or "b2-b3" deletions), particularly in infertile patients. DAZ genes are present in 4 copies and rearrangements involve a modification of the number of DAZ genes.<h4>Methods</h4>In addition to STS plus/minus PCR, we developed a quantitative tec ...[more]

PMID: 17410421

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Project description:Recent research has indicated that fungal biopesticides could augment existing malaria vector control tools. Here we present a set of methodologies to monitor the in vivo kinetics of entomopathogenic fungi in Anopheles in the presence or absence of malaria parasites using quantitative real-time PCR. Three qPCR assays were successfully developed for counting fungal genomes: "specific" assays capable of distinguishing two well characterized fungal entomopathogens Beauveria bassiana isolate IMI391510 and Metarhizium anisopliae var. acridum isolate IMI330189, both of which have previously been shown to be virulent to Anopheles mosquitoes, and a "generic" fungal assay for determining any fungal burden. A fourth assay to Plasmodium chabaudi enabled quantification of co-infecting malarial parasites. All qPCR assays provide sensitive, target-specific, and robust quantification over a linear range of greater than five orders of magnitude (seven orders of magnitude for the fungal assays). B. bassiana growth within mosquitoes exposed to three different conidial challenge doses was monitored using the B. bassiana-specific assay and represents the first description of entomopathogenic fungal replication within an insect host. This revealed that, irrespective of challenge dose, after several days of relatively little replication, a sudden on-set of substantial nuclear division occurs, accompanied by physical fungal growth (hyphae) within the mosquito haemocoel shortly before death. Exposure to higher densities of conidia resulted in significantly greater pick-up by mosquitoes and to elevated fungal burdens at each time point sampled. High fungal burdens, comparable to those identified in cadavers, were attained more rapidly and mortalities occurred earlier post-exposure with increasing challenge dose. The lines of research made possible by the qPCR assays described here will contribute to optimization of fungal biopesticides against malaria and other vector-borne diseases.

Dataset Information

Quantitative PCR technique for the identification of microrearrangements of the AZFc region.

Publications

Quantitative PCR technique for the identification of microrearrangements of the AZFc region.

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