A total of subjects, belonging to 14 affected families, were genotyped to confirm the concordance with the singleplex method used previously. Conclusions: This is a useful strategy that, together with automated computer-based allele detection, allows reliable, simple, faster, and cheaper genetic analysis than the previous singleplex method..
Autosomal dominant polycystic kidney disease ADPKD is the third most common cause of chronic kidney disease 1 and the most life-threatening hereditary disease. To date, two genes that cause the disease have been identified: PKD1, in chromosome region 16p ADPKD is easily diagnosed with ultrasound when cysts are already present.
However, genetic analysis can be used when ultrasound results are unclear or when a definitive diagnosis is needed for a patient younger than Clinical trials are currently being carried out to test the ability of some drugs to halt the progression of the disease.
Molecular analysis can be done either through a direct search for mutations, or indirectly through gene binding analysis, using informative markers microsatellites located near the genes that interest us. These markers allow us to track the chromosome responsible for the disease through generations of the family being studied.
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The suitability of these markers for our population was studied in 30 affected families. The sensitivity and specificity of the genetic analysis were A total of individuals, 52 affected by the disease These individuals belonged to 14 families with ADPKD who were randomly selected from those that were included in the preliminary analysis. Furthermore, we obtained informed consent from all participants in the study before they were included in it.. A new series of primers was designed for developing the multiplex-PCR, which ensured equivalent amplification efficiency for all DNA fragments, as described previously.
As a primer-binding site, complex sequences, homopolymers with more than five bases and potentially stable secondary structures below the threshold of kcal mol-1 of free energy in order to guarantee the sensitivity and specificity of the multiplex amplification. To differentiate amplified products of similar sizes, we used a multi-colour fluorescent and marked the clockwise primer for each pair with one of the following fluorophores: 6- FAM, HEX or NED.
The quantity of each primer included in the final mixture was established empirically, in accordance with results from consecutive tests. Furthermore, different series dilutions were tested for MgCl2, between 1. A product of the desired size was obtained for all loci.
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In order to analyse the robustness of multiplex-PCR, duplicate analyses were run for each sample.. We used the software GeneScan Analysis 3. Initially, four markers D4S, CW2, D16S and SM7 gave off a weak signal, which suggested poor amplification under the same conditions and with the same quantities of primer. Reactions improved when primer quantities increased, thus reaching the desired balance between signals. The best results were obtained with a concentration of 3mM MgCl2 for all markers figure 2.
Slightly higher concentrations inhibited amplification.. Markers were completely informative for all patients and in Multiplex-PCR demonstrated a specificity of To develop this technique, it was necessary to run nine different PCR reactions for each individual, mix the amplification products, and lastly, prepare the sample for capillary electrophoresis.
An important part of our work consisted of exploring various aspects of multiplex-PCR methods. Primers were designed paying special attention to avoid self-alignment and dimer formation, obtaining maximum efficiency and balance between the quantities of amplification products. The primers were subjected to HPLC in order to obtain pure, homogeneous molecules, without a surplus of fluorophores that might interfere with allele detection. Genetic analysis using the multiplex-PCR system affords greater ease in both genetic counselling and early diagnosis, which is increasingly important for this disease.
To conclude, this strategy, in conjunction with automatic allele detection, allows us to carry out a reliable, simple genetic analysis that is faster and less expensive than the one based on individual amplifications of microsatellites, which encourages including family studies in the clinical routine.. Home Articles in press Archive. ISSN: Previous article Next article. August Pages DOI: Genetic diagnosis of autosomal dominant polycystic kidney disease using multiplex-PCR.
Molecular Methods for Diagnosis of Genetic Diseases
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Molecular diagnosis of genetic disease.
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New Molecular Diagnosis Approaches — From the Identification of Mutations to their Characterization
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Actions Shares. Embeds 0 No embeds. No notes for slide. Molecular Methods for Diagnosis of Genetic Diseases 1. Professor of Genetics 2. Old basic tedious and time consuming experiments. Rapid and high throughput in a narrow spaced laboratory. Southern blotting 2. Restriction fragment length polymorphism RFLP 3. Heteroduplex migration analysis 6.
Single- strand conformational polymorphism SSCP analysis.
Denaturating gradient gel electrophoresis DGGE 8. Conformation-sensitive capillary electrophoresis CSCE Oligonucleotide ligation assay OLA Real-time PCR High-resolution melt curve analysis HRM Restrictionfragment SouthernBlotting 7. The PCR products are run on a special denaturing gradient gel that contains increasing concentrations of a denaturing chemical e. PCR products are injected in a column containing an increasing gradient of mobile phase acetonitrite required to elute each homo- or heteroduplex.
Its detection capability is dependant upon fragment length, sequence, mutation, PCR quality and analytical equipment.