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Effect of co-axially hybridized gene targets on hybridization efficiency of microarrayed DNA probes.

ABSTRACT: The effect of relative size of two co-axially hybridized gene targets on the hybridization efficiency was studied for two DNA probe configurations and various probe concentrations. Each of two sets of microarrayed probes contained a pair of DNA probes and a pair of their complementary samples labeled with two distinct fluorescent dyes. The sequence of each probe is especially designed so that two targets are simultaneously complementary to two adjacent sections of the probe. The molecular steric effect on the hybridization efficiency is investigated by comparing the dye signals between configurations of one-target and two-target hybridization scenarios. The results show that a low probe concentration gives better hybridization efficiency and the first-hybridization conducted by a shorter-size DNA target improves the hybridization efficiency of the second target coupling onto the same probe.

SUBMITTER: Jiang KR

PROVIDER: S-EPMC7185593 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Effect of co-axially hybridized gene targets on hybridization efficiency of microarrayed DNA probes.

Jiang Kai Ren KR Huang Jie-Len JL Chen Chia-Chun CC Su Hung-Ju HJ Wu Jui-Chuang JC

Journal of the Taiwan Institute of Chemical Engineers 20101030 1

The effect of relative size of two co-axially hybridized gene targets on the hybridization efficiency was studied for two DNA probe configurations and various probe concentrations. Each of two sets of microarrayed probes contained a pair of DNA probes and a pair of their complementary samples labeled with two distinct fluorescent dyes. The sequence of each probe is especially designed so that two targets are simultaneously complementary to two adjacent sections of the probe. The molecular steric ...[more]

PMID: 32362954

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Project description:BackgroundToxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid.MethodsTwo separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.ResultsComparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.ConclusionWe have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Dataset Information

Effect of co-axially hybridized gene targets on hybridization efficiency of microarrayed DNA probes.

Publications

Effect of co-axially hybridized gene targets on hybridization efficiency of microarrayed DNA probes.

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