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of all cases&#41;&#44; and PKD2 in chromosome region 4q21 &#40;15&#37; of all cases&#41;&#46; ADPKD is easily diagnosed with ultrasound when cysts are already present&#46; However&#44; genetic analysis can be used when ultrasound results are unclear or when a definitive diagnosis is needed for a patient younger than 30&#46; An important use of molecular diagnosis is genetic counselling&#44; which is the practice of providing the patient with information about his&#47;her disease&#44; the inheritance pattern and the risk of transmitting it to descendents &#40;50&#37;&#41;&#46; It can also be used in families with ADPKD to confirm potential kidney donors&#46; Clinical trials are currently being carried out to test the ability of some drugs to halt the progression of the disease&#46;<span class="elsevierStyleSup">2</span> Patients who are diagnosed early may be candidates for treatment with these new agents in the future&#46;</p><p class="elsevierStylePara">Molecular analysis can be done either through a direct search for mutations&#44; or indirectly through gene binding analysis&#44; using informative markers &#40;microsatellites&#41; located near the genes that interest us&#46; These markers allow us to track the chromosome responsible for the disease through generations of the family being studied&#46; Two microsatellite series selected in a preliminary study for PKD1 &#40;D16S521&#44; KG8&#44; AC2&#46;5&#44; CW2&#44; SM7&#41; and PKD2 &#40;D4S1538&#44; D4S1534&#44; D4S423&#44; D4S414&#41; were used for the gene binding analysis in families with ADPKD &#40;figure 1&#41;&#46; The suitability of these markers for our population was studied in 30 affected families&#46;<span class="elsevierStyleSup">4</span> The markers were amplified individually and subsequently mixed for the analysis&#44; which permitted unequivocal genetic identification of 97&#46;7&#37; in patients and 88&#46;7&#37; in healthy individuals&#46; The sensitivity and specificity of the genetic analysis were 90&#46;7&#37; &#40;CI 95&#37;&#44; 85&#46;7-95&#46;7&#41; and 86&#46;8&#37; &#40;CI 95&#37;&#44; 80&#46;6-93&#46;0&#41; respectively&#46;</p><p class="elsevierStylePara">In this study&#44; we present a new analysis of PKD1 and PKD2 microsatellites based on multiplex-PCR and capillary electrophoresis&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">MATERIAL AND METHOD</span></p><p class="elsevierStylePara">A total of 110 individuals&#44; 52 affected by the disease &#40;57&#46;7&#37; male&#41; and 58 not affected &#40;39&#46;7&#37; male&#41; were analysed using the new multiplex-PCR system&#46; These individuals belonged to 14 families with ADPKD who were randomly selected from those that were included in the preliminary analysis&#46;<span class="elsevierStyleSup">4</span> The study was approved by the Ethics Committee at Dr Negr&#237;n University Hospital of Gran Canaria&#46; Furthermore&#44; we obtained informed consent from all participants in the study before they were included in it&#46;</p><p class="elsevierStylePara">A new series of primers was designed for developing the multiplex-PCR&#44; which ensured equivalent amplification efficiency for all DNA fragments&#44; as described previously&#46;<span class="elsevierStyleSup">5</span> The length of the amplified fragments varied between 75 and 158 base pairs &#40;table 1&#41;&#46; The objective was to obtain primers with a GC content of about 35-60&#37; and a theoretical melting temperature of 60 &#177; 2&#8747; C in a salt concentration &#40;K&#43;&#44; Na&#43;&#44; Tris&#43; or NH4&#43;&#41; of 180mM&#46; As a primer-binding site&#44; complex sequences&#44; homopolymers with more than five bases and potentially stable secondary structures &#40;below the threshold of -13kcal mol-1 of free energy&#41; in order to guarantee the sensitivity and specificity of the multiplex amplification&#46;<span class="elsevierStyleSup">6</span> We used the program available at Integrated DNA Technologies <a href="http&#58;&#47;&#47;www&#46;idtdna&#46;com" class="elsevierStyleCrossRefs">&#40;http&#58;&#47;&#47;www&#46;idtdna&#46;com</a>&#47;Scitools&#47;Applications&#47;mFold&#47;&#41; to verify that none of the candidate strains presented secondary structures or hairpins&#46; The strains were synthesised and purified using HPLC by Bioron GmbH &#40;Ludwigshafen <br></br>Germany&#41; and Applied Biosystems &#40;Foster City&#44; U&#46;S&#46;A&#41;&#46; To differentiate amplified products of similar sizes&#44; we used a multi-colour fluorescent and marked the clockwise primer for each pair with one of the following fluorophores&#58; 6- FAM&#44; HEX or NED&#46; Each pair of primers was tested in an individual PCR with five DNA samples&#44; under identical amplification conditions&#44; before carrying out the multiplex PCR reaction&#46; The quantity of each primer included in the final mixture was established empirically&#44; in accordance with results from consecutive tests&#46; Furthermore&#44; different series dilutions were tested for MgCl2&#44; between 1&#46;5 and 5mM&#44; in order to obtain the maximum amplification efficiency&#46;</p><p class="elsevierStylePara">Owing to a slight overlap between the size ranges for CW2 and D4S414 in some samples&#44; we decided to analyse PKD1 and PKD2 markers separately&#46; Both reactions contained 1-10ng of DNA&#44; 3mM MgCl2&#44; 200&#956;M of each dNTP&#44; 0&#46;05-0&#46;1&#956;M of each primer and 0&#46;5U AmpliTaq Gold polymerase &#40;Applied Biosystems&#41; in a final reaction volume of 12&#46;5&#956;l&#46; The amplification protocol was carried out in a GeneAmp PCR System 9700 Thermal Cycler &#40;Applied Biosystems&#41;&#44; and consisted of denaturalisation at 95&#8747; C during 5 minutes&#44; followed by 26 cycles of 95&#8747; C during 30 seconds&#44; 65&#8747; C during 30 seconds and 72&#8747; C during 45 seconds&#44; with a final extension step of 20 minutes at 72&#8747; C&#46; A product of the desired size was obtained for all loci&#46; In order to analyse the robustness of multiplex-PCR&#44; duplicate analyses were run for each sample&#46;</p><p class="elsevierStylePara">Detection of PCR fragments using capillary electrophoresis was carried out in an ABI TM 310 sequencer &#40;Applied Biosystems&#41;&#44; using an uncovered capillary 47cm long and POP4 polymer &#40;Applied Biosystems&#41;&#46; For the electrophoresis&#44; 1&#956;l of each PCR product was mixed with 20&#956;l de-ionised formamide &#40;Applied Biosystems&#41; and 0&#46;2&#956;l of GeneScan-ROX molecular weight marker &#40;Applied Biosystems&#41;&#46; Samples were injected with 15kV during 5 seconds&#44; and separated with the same voltage and a temperature of 60&#8747; C during 24 minutes&#46;We used the software GeneScan Analysis 3&#46;1&#46;2 &#40;Applied Biosystems&#41; to estimate fragment size&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">RESULTS</span></p><p class="elsevierStylePara">Initially&#44; four markers &#40;D4S423&#44; CW2&#44; D16S521 and SM7&#41; gave off a weak signal&#44; which suggested poor amplification under the same conditions and with the same quantities of primer&#46; Reactions improved when primer quantities increased&#44; thus reaching the desired balance between signals&#46; The best results were obtained with a concentration of 3mM MgCl2 for all markers &#40;figure 2&#41;&#46; Slightly higher concentrations inhibited amplification&#46;</p><p class="elsevierStylePara">Identification of individual haplotypes showed 100&#37; concordance with the single-PCR results gathered prior to the analysis&#46; Markers were completely informative for all patients and in 91&#46;4&#37; of the healthy individuals&#46; Multiplex-PCR demonstrated a specificity of 88&#46;5&#37; &#40;CI 95&#37;&#44; 75&#46;9-95&#46;2&#41; and a sensitivity of 87&#46;9 &#40;CI 95&#37;&#44; 76&#46;1-94&#46;6&#41;&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">DISCUSSION</span></p><p class="elsevierStylePara">A large number of markers for analysing the PKD1 and PKD2 genes have been described&#46; In our preliminary study&#44;<span class="elsevierStyleSup">4</span> we used a simple PCR system to amplify two series of microsatellite PKD1 and PKD2 markers&#46; To develop this technique&#44; it was necessary to run nine different PCR reactions for each individual&#44; mix the amplification products&#44; and lastly&#44; prepare the sample for capillary electrophoresis&#46; Until now&#44; the simplest multiplex-PCR system published for ADPKD analysis was the one by Vouk et al&#44;<span class="elsevierStyleSup">7</span> which consists of three amplification reactions&#44; combining PKD1 and PKD2 markers&#46;</p><p class="elsevierStylePara">An important part of our work consisted of exploring various aspects of multiplex-PCR methods&#46; Primers were designed paying special attention to avoid self-alignment and dimer formation&#44; obtaining maximum efficiency and balance between the quantities of amplification products&#46; The primers were subjected to HPLC in order to obtain pure&#44; homogeneous molecules&#44; without a surplus of fluorophores that might interfere with allele detection&#46; To avoid non-specific amplification products&#44; we used the PCR programme &#8220;Hot Start&#8221; &#40;in which the polymerase is activated at high temperatures and the PCR products accumulate slowly&#41;&#46;</p><p class="elsevierStylePara">We have developed a new multiplex-PCR system for genotyping nine microsatellite ADPKD markers in only two reactions&#46; An additional advantage of this system is that it permits independent analysis of PKD1 and PKD2&#46; Given that 85-90&#37; of ADPKD cases are linked to PKD1&#44; analysing the markers for this gene will be sufficient in order to determine the family&#8217;s genetic characteristics in many cases&#46; Genetic analysis using the multiplex-PCR system affords greater ease in both genetic counselling and early diagnosis&#44; which is increasingly important for this disease&#46; To conclude&#44; this strategy&#44; in conjunction with automatic allele detection&#44; allows us to carry out a reliable&#44; simple genetic analysis that is faster and less expensive than the one based on individual amplifications of microsatellites&#44; which encourages including family studies in the clinical routine&#46;</p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;f1&#95;328&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_f1_328.jpg"></img></a></p><p class="elsevierStylePara">Figure 1&#46; </p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;t1&#95;329&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_t1_329.jpg" alt="Microsatellites and primers"></img></a></p><p class="elsevierStylePara">Table 1&#46; Microsatellites and primers</p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;f2&#95;330&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_f2_330.jpg"></img></a></p><p class="elsevierStylePara">Figure 2&#46; </p>"
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        "resumen" => "<p class="elsevierStylePara">Introducci&#243;n&#58; La poliquistosis renal autos&#243;mica dominante &#40;PQRAD&#41; es la enfermedad hereditaria m&#225;s frecuente con mayor amenaza para la vida&#46; El an&#225;lisis molecular de microsat&#233;lites polim&#243;rficos&#44; localizados en torno a los genes responsables de la enfermedad &#40;PKD1 y PKD2&#41; se utiliza para confirmar el diagn&#243;stico y dar consejo gen&#233;tico&#46; M&#233;todos&#58; Se desarroll&#243; un m&#233;todo de genotipado para cinco marcadores de PKD1 y cuatro de PKD2&#44; basado en dos reacciones de PCR m&#250;ltiple y an&#225;lisis por electroforesis capilar&#46; Un total de 110 individuos&#44; pertenecientes a 14 familias afectas&#44; fueron genotipados para confirmar la concordancia con el m&#233;todo de PCR simple usado previamente&#46; Resultados&#58; El rango de tama&#241;o de los fragmentos amplificados fue desde 95 a 154 pb&#44; y los perfiles completos de marcadores microsat&#233;lites se obtuvieron a partir de 1-5 ng de ADN&#46; La especificidad de la PCR m&#250;ltiple fue del 88&#44;5&#37; &#40;IC 95&#37; 75&#44;9-95&#44;2&#41;&#44; y la sensibilidad del 87&#44;9 &#40;IC 95&#37; 76&#44;1-94&#44;6&#41;&#46; Conclusiones&#58; Esta estrategia&#44; junto con la detecci&#243;n autom&#225;tica de alelos&#44; permite realizar un an&#225;lisis gen&#233;tico fiable&#44; sencillo&#44; m&#225;s r&#225;pido y de menor coste que el basado en amplificaciones individuales de los microsat&#233;lites&#46;</p>"
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Genetic diagnosis of autosomal dominant polycystic kidney disease using multiplex-PCR
Diagnóstico Genético de Poliquistosis Renal Autosómica Dominante mediante PCR múltiple1
Nisa Buset Ríosa, María J. Torres Galvána, Juan J. Sánchezb, Carmen R. Hernándezc, Érika Hernándeza, José C. Rodríguez Pérezd
a Unidad de Investigación, Hospital Universitario de Gran Canaria Dr. Negrín, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria Las Palmas de Gran Canaria España,
b Instituto Nacional de Toxicología y Ciencias Forenses, Santa Cruz de Tenerife, Santa Cruz de Tenerife, España,
c Servicio de Radiodiagnóstico, Hospital Universitario de Gran Canaria Dr. Negrín, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria Las Palmas de Gran Canaria España,
d Unidad de Investigación y 2Servicio de Nefrología, Hospital Universitario de Gran Canaria Dr. Negrín, Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria Las Palmas de Gran Canaria España,
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    "textoCompleto" => "<p class="elsevierStylePara"><span class="elsevierStyleBold">INTRODUCTION</span></p><p class="elsevierStylePara">Autosomal dominant polycystic kidney disease &#40;ADPKD&#41; is the third most common cause of chronic kidney disease<span class="elsevierStyleSup">1</span> and the most life-threatening hereditary disease&#46;<span class="elsevierStyleSup">2</span> Its prevalence varies between 1&#47;400 and 1&#47;1000 individuals&#44; depending on the population&#46;<span class="elsevierStyleSup">3</span> It is characterised by the formation of multiple cysts in the kidneys&#44; and by cardiovascular and gastrointestinal disorders such as intracranial or aortic aneurysm&#44; mitral valve prolapse&#44; hernia of the abdominal wall&#44; etc&#46; 50&#37; of these patients develop chronic kidney disease during their fifth decade&#46;</p><p class="elsevierStylePara">To date&#44; two genes that cause the disease have been identified&#58; PKD1&#44; in chromosome region 16p13&#46;3 &#40;85&#37; of all cases&#41;&#44; and PKD2 in chromosome region 4q21 &#40;15&#37; of all cases&#41;&#46; ADPKD is easily diagnosed with ultrasound when cysts are already present&#46; However&#44; genetic analysis can be used when ultrasound results are unclear or when a definitive diagnosis is needed for a patient younger than 30&#46; An important use of molecular diagnosis is genetic counselling&#44; which is the practice of providing the patient with information about his&#47;her disease&#44; the inheritance pattern and the risk of transmitting it to descendents &#40;50&#37;&#41;&#46; It can also be used in families with ADPKD to confirm potential kidney donors&#46; Clinical trials are currently being carried out to test the ability of some drugs to halt the progression of the disease&#46;<span class="elsevierStyleSup">2</span> Patients who are diagnosed early may be candidates for treatment with these new agents in the future&#46;</p><p class="elsevierStylePara">Molecular analysis can be done either through a direct search for mutations&#44; or indirectly through gene binding analysis&#44; using informative markers &#40;microsatellites&#41; located near the genes that interest us&#46; These markers allow us to track the chromosome responsible for the disease through generations of the family being studied&#46; Two microsatellite series selected in a preliminary study for PKD1 &#40;D16S521&#44; KG8&#44; AC2&#46;5&#44; CW2&#44; SM7&#41; and PKD2 &#40;D4S1538&#44; D4S1534&#44; D4S423&#44; D4S414&#41; were used for the gene binding analysis in families with ADPKD &#40;figure 1&#41;&#46; The suitability of these markers for our population was studied in 30 affected families&#46;<span class="elsevierStyleSup">4</span> The markers were amplified individually and subsequently mixed for the analysis&#44; which permitted unequivocal genetic identification of 97&#46;7&#37; in patients and 88&#46;7&#37; in healthy individuals&#46; The sensitivity and specificity of the genetic analysis were 90&#46;7&#37; &#40;CI 95&#37;&#44; 85&#46;7-95&#46;7&#41; and 86&#46;8&#37; &#40;CI 95&#37;&#44; 80&#46;6-93&#46;0&#41; respectively&#46;</p><p class="elsevierStylePara">In this study&#44; we present a new analysis of PKD1 and PKD2 microsatellites based on multiplex-PCR and capillary electrophoresis&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">MATERIAL AND METHOD</span></p><p class="elsevierStylePara">A total of 110 individuals&#44; 52 affected by the disease &#40;57&#46;7&#37; male&#41; and 58 not affected &#40;39&#46;7&#37; male&#41; were analysed using the new multiplex-PCR system&#46; These individuals belonged to 14 families with ADPKD who were randomly selected from those that were included in the preliminary analysis&#46;<span class="elsevierStyleSup">4</span> The study was approved by the Ethics Committee at Dr Negr&#237;n University Hospital of Gran Canaria&#46; Furthermore&#44; we obtained informed consent from all participants in the study before they were included in it&#46;</p><p class="elsevierStylePara">A new series of primers was designed for developing the multiplex-PCR&#44; which ensured equivalent amplification efficiency for all DNA fragments&#44; as described previously&#46;<span class="elsevierStyleSup">5</span> The length of the amplified fragments varied between 75 and 158 base pairs &#40;table 1&#41;&#46; The objective was to obtain primers with a GC content of about 35-60&#37; and a theoretical melting temperature of 60 &#177; 2&#8747; C in a salt concentration &#40;K&#43;&#44; Na&#43;&#44; Tris&#43; or NH4&#43;&#41; of 180mM&#46; As a primer-binding site&#44; complex sequences&#44; homopolymers with more than five bases and potentially stable secondary structures &#40;below the threshold of -13kcal mol-1 of free energy&#41; in order to guarantee the sensitivity and specificity of the multiplex amplification&#46;<span class="elsevierStyleSup">6</span> We used the program available at Integrated DNA Technologies <a href="http&#58;&#47;&#47;www&#46;idtdna&#46;com" class="elsevierStyleCrossRefs">&#40;http&#58;&#47;&#47;www&#46;idtdna&#46;com</a>&#47;Scitools&#47;Applications&#47;mFold&#47;&#41; to verify that none of the candidate strains presented secondary structures or hairpins&#46; The strains were synthesised and purified using HPLC by Bioron GmbH &#40;Ludwigshafen <br></br>Germany&#41; and Applied Biosystems &#40;Foster City&#44; U&#46;S&#46;A&#41;&#46; To differentiate amplified products of similar sizes&#44; we used a multi-colour fluorescent and marked the clockwise primer for each pair with one of the following fluorophores&#58; 6- FAM&#44; HEX or NED&#46; Each pair of primers was tested in an individual PCR with five DNA samples&#44; under identical amplification conditions&#44; before carrying out the multiplex PCR reaction&#46; The quantity of each primer included in the final mixture was established empirically&#44; in accordance with results from consecutive tests&#46; Furthermore&#44; different series dilutions were tested for MgCl2&#44; between 1&#46;5 and 5mM&#44; in order to obtain the maximum amplification efficiency&#46;</p><p class="elsevierStylePara">Owing to a slight overlap between the size ranges for CW2 and D4S414 in some samples&#44; we decided to analyse PKD1 and PKD2 markers separately&#46; Both reactions contained 1-10ng of DNA&#44; 3mM MgCl2&#44; 200&#956;M of each dNTP&#44; 0&#46;05-0&#46;1&#956;M of each primer and 0&#46;5U AmpliTaq Gold polymerase &#40;Applied Biosystems&#41; in a final reaction volume of 12&#46;5&#956;l&#46; The amplification protocol was carried out in a GeneAmp PCR System 9700 Thermal Cycler &#40;Applied Biosystems&#41;&#44; and consisted of denaturalisation at 95&#8747; C during 5 minutes&#44; followed by 26 cycles of 95&#8747; C during 30 seconds&#44; 65&#8747; C during 30 seconds and 72&#8747; C during 45 seconds&#44; with a final extension step of 20 minutes at 72&#8747; C&#46; A product of the desired size was obtained for all loci&#46; In order to analyse the robustness of multiplex-PCR&#44; duplicate analyses were run for each sample&#46;</p><p class="elsevierStylePara">Detection of PCR fragments using capillary electrophoresis was carried out in an ABI TM 310 sequencer &#40;Applied Biosystems&#41;&#44; using an uncovered capillary 47cm long and POP4 polymer &#40;Applied Biosystems&#41;&#46; For the electrophoresis&#44; 1&#956;l of each PCR product was mixed with 20&#956;l de-ionised formamide &#40;Applied Biosystems&#41; and 0&#46;2&#956;l of GeneScan-ROX molecular weight marker &#40;Applied Biosystems&#41;&#46; Samples were injected with 15kV during 5 seconds&#44; and separated with the same voltage and a temperature of 60&#8747; C during 24 minutes&#46;We used the software GeneScan Analysis 3&#46;1&#46;2 &#40;Applied Biosystems&#41; to estimate fragment size&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">RESULTS</span></p><p class="elsevierStylePara">Initially&#44; four markers &#40;D4S423&#44; CW2&#44; D16S521 and SM7&#41; gave off a weak signal&#44; which suggested poor amplification under the same conditions and with the same quantities of primer&#46; Reactions improved when primer quantities increased&#44; thus reaching the desired balance between signals&#46; The best results were obtained with a concentration of 3mM MgCl2 for all markers &#40;figure 2&#41;&#46; Slightly higher concentrations inhibited amplification&#46;</p><p class="elsevierStylePara">Identification of individual haplotypes showed 100&#37; concordance with the single-PCR results gathered prior to the analysis&#46; Markers were completely informative for all patients and in 91&#46;4&#37; of the healthy individuals&#46; Multiplex-PCR demonstrated a specificity of 88&#46;5&#37; &#40;CI 95&#37;&#44; 75&#46;9-95&#46;2&#41; and a sensitivity of 87&#46;9 &#40;CI 95&#37;&#44; 76&#46;1-94&#46;6&#41;&#46;<span class="elsevierStyleBold"></span></p><p class="elsevierStylePara"><span class="elsevierStyleBold">DISCUSSION</span></p><p class="elsevierStylePara">A large number of markers for analysing the PKD1 and PKD2 genes have been described&#46; In our preliminary study&#44;<span class="elsevierStyleSup">4</span> we used a simple PCR system to amplify two series of microsatellite PKD1 and PKD2 markers&#46; To develop this technique&#44; it was necessary to run nine different PCR reactions for each individual&#44; mix the amplification products&#44; and lastly&#44; prepare the sample for capillary electrophoresis&#46; Until now&#44; the simplest multiplex-PCR system published for ADPKD analysis was the one by Vouk et al&#44;<span class="elsevierStyleSup">7</span> which consists of three amplification reactions&#44; combining PKD1 and PKD2 markers&#46;</p><p class="elsevierStylePara">An important part of our work consisted of exploring various aspects of multiplex-PCR methods&#46; Primers were designed paying special attention to avoid self-alignment and dimer formation&#44; obtaining maximum efficiency and balance between the quantities of amplification products&#46; The primers were subjected to HPLC in order to obtain pure&#44; homogeneous molecules&#44; without a surplus of fluorophores that might interfere with allele detection&#46; To avoid non-specific amplification products&#44; we used the PCR programme &#8220;Hot Start&#8221; &#40;in which the polymerase is activated at high temperatures and the PCR products accumulate slowly&#41;&#46;</p><p class="elsevierStylePara">We have developed a new multiplex-PCR system for genotyping nine microsatellite ADPKD markers in only two reactions&#46; An additional advantage of this system is that it permits independent analysis of PKD1 and PKD2&#46; Given that 85-90&#37; of ADPKD cases are linked to PKD1&#44; analysing the markers for this gene will be sufficient in order to determine the family&#8217;s genetic characteristics in many cases&#46; Genetic analysis using the multiplex-PCR system affords greater ease in both genetic counselling and early diagnosis&#44; which is increasingly important for this disease&#46; To conclude&#44; this strategy&#44; in conjunction with automatic allele detection&#44; allows us to carry out a reliable&#44; simple genetic analysis that is faster and less expensive than the one based on individual amplifications of microsatellites&#44; which encourages including family studies in the clinical routine&#46;</p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;f1&#95;328&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_f1_328.jpg"></img></a></p><p class="elsevierStylePara">Figure 1&#46; </p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;t1&#95;329&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_t1_329.jpg" alt="Microsatellites and primers"></img></a></p><p class="elsevierStylePara">Table 1&#46; Microsatellites and primers</p><p class="elsevierStylePara"><a href="grande&#47;11618078&#95;f2&#95;330&#46;jpg" class="elsevierStyleCrossRefs"><img src="11618078_f2_330.jpg"></img></a></p><p class="elsevierStylePara">Figure 2&#46; </p>"
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        "resumen" => "<p class="elsevierStylePara">Introducci&#243;n&#58; La poliquistosis renal autos&#243;mica dominante &#40;PQRAD&#41; es la enfermedad hereditaria m&#225;s frecuente con mayor amenaza para la vida&#46; El an&#225;lisis molecular de microsat&#233;lites polim&#243;rficos&#44; localizados en torno a los genes responsables de la enfermedad &#40;PKD1 y PKD2&#41; se utiliza para confirmar el diagn&#243;stico y dar consejo gen&#233;tico&#46; M&#233;todos&#58; Se desarroll&#243; un m&#233;todo de genotipado para cinco marcadores de PKD1 y cuatro de PKD2&#44; basado en dos reacciones de PCR m&#250;ltiple y an&#225;lisis por electroforesis capilar&#46; Un total de 110 individuos&#44; pertenecientes a 14 familias afectas&#44; fueron genotipados para confirmar la concordancia con el m&#233;todo de PCR simple usado previamente&#46; Resultados&#58; El rango de tama&#241;o de los fragmentos amplificados fue desde 95 a 154 pb&#44; y los perfiles completos de marcadores microsat&#233;lites se obtuvieron a partir de 1-5 ng de ADN&#46; La especificidad de la PCR m&#250;ltiple fue del 88&#44;5&#37; &#40;IC 95&#37; 75&#44;9-95&#44;2&#41;&#44; y la sensibilidad del 87&#44;9 &#40;IC 95&#37; 76&#44;1-94&#44;6&#41;&#46; Conclusiones&#58; Esta estrategia&#44; junto con la detecci&#243;n autom&#225;tica de alelos&#44; permite realizar un an&#225;lisis gen&#233;tico fiable&#44; sencillo&#44; m&#225;s r&#225;pido y de menor coste que el basado en amplificaciones individuales de los microsat&#233;lites&#46;</p>"
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        "resumen" => "<p class="elsevierStylePara">Background&#58; Autosomal Dominant Polycystic Kidney Disease &#40;ADPKD&#41; is the most common life-threatening hereditary disease&#46; Molecular analysis with highly polymorphic short tandem repeats&#44; located in the vicinity of the two genes responsible for the disease &#40;PKD1 and PKD2&#41;&#44; is used to confirm diagnosis and give genetic counseling to members of affected families&#46; Methods&#58; We have developed a new assay to genotype five PKD1 and four PKD2 markers&#44; based on two multiplex PCR reactions&#44; and capillary electrophoresis analysis&#46; A total of 110 subjects&#44; belonging to 14 affected families&#44; were genotyped to confirm the concordance with the singleplex method used previously&#46; Results&#58; The amplicons ranged from 95 to 154 bp in length&#44; and complete STR profiles were obtained from 1-5 ng DNA&#46; The specificity of the multiplex PCR system was 88&#44;5&#37; &#40;95&#37;CI&#61; 75&#44;9-95&#44;2&#41;&#44; and the sensitivity&#44; 87&#44;9 &#40;95&#37;CI&#61; 76&#44;1-94&#44;6&#41;&#46; Conclusions&#58; This is a useful strategy that&#44; together with automated computer-based allele detection&#44; allows reliable&#44; simple&#44; faster&#44; and cheaper genetic analysis than the previous singleplex method&#46;</p>"
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ISSN: 20132514
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