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Digital polymeraskedjereaktion (digital PC … Digital polymeraskedjereaktion (digital PCR, dPCR, ddPCR, cdPCR) är en variant av den traditionella polymeraskedjereaktionen (PCR). Metoden används för att detektera och kvantifiera nukleinsyra (exempelvis DNA och RNA, för det sistnämnda används digital RT-PCR). Till skillnad från kvantitativ realtids PCR (qPCR) sker kvantifiering av templatmolekyler direkt i varje prov. Detta gör att externa kalibreringskurvor (kvantifieringsstandarder) inte är nödvändiga. En annan viktig skillnad mot qPCR är att detektion och kvantifiering sker efter att PCR-reaktionen är genomförd (så kallad end-point detektion), istället för under reaktionens gång som i qPCR (realtidsdetektion). Grundprincipen för digital PCR är att varje prov (innehållande templat, enzymmix, målspecifika primrar och hydrolysprober eller interkalerande färg) delas upp i tusentals små, individuella partitioner. Varje partition utgör en separat reaktion. De templatmolekyler som finns i provet fördelar sig slumpmässigt i dessa partitioner, vilket innebär att vissa partitioner kommer att innehålla en eller flera målmolekyler (”1”) och andra kommer att sakna målmolekyler (”0”). Efter uppdelningen körs PCR i en traditionell termocykler. Amplifiering sker enbart i de partitioner som innehåller minst en templatmolekyl. Efter att PCR genomförts kommer positiva partitioner (som innehåller minst en templatmolekyl) ha högre fluorescensintensitet jämfört med negativa partitioner. Antalet positiva och negativa partitioner räknas och beräkning av den ursprungliga mängden templatmolekyler sker med hjälp av Poissonstatistik. Metoden kräver att minst en partition saknar templat (det vill säga ger ett negativt resultat). Är alla partitioner positiva måste provet spädas och analyseras om.iva måste provet spädas och analyseras om.
, Die Digital Polymerase Chain Reaction (zu deutsch ‚Digitale Polymerase-Kettenreaktion‘, auch dPCR, digital PCR, Digital-PCR) ist eine biochemische Methode zur Mengenbestimmung einzelner DNA-Sequenzen. Sie ist eine Variante der Polymerase-Kettenreaktion.
, Digital polymerase chain reaction (digital … Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for next-generation sequencing.of samples for next-generation sequencing.
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Digital polymeraskedjereaktion (digital PC … Digital polymeraskedjereaktion (digital PCR, dPCR, ddPCR, cdPCR) är en variant av den traditionella polymeraskedjereaktionen (PCR). Metoden används för att detektera och kvantifiera nukleinsyra (exempelvis DNA och RNA, för det sistnämnda används digital RT-PCR). Till skillnad från kvantitativ realtids PCR (qPCR) sker kvantifiering av templatmolekyler direkt i varje prov. Detta gör att externa kalibreringskurvor (kvantifieringsstandarder) inte är nödvändiga. En annan viktig skillnad mot qPCR är att detektion och kvantifiering sker efter att PCR-reaktionen är genomförd (så kallad end-point detektion), istället för under reaktionens gång som i qPCR (realtidsdetektion).onens gång som i qPCR (realtidsdetektion).
, Die Digital Polymerase Chain Reaction (zu deutsch ‚Digitale Polymerase-Kettenreaktion‘, auch dPCR, digital PCR, Digital-PCR) ist eine biochemische Methode zur Mengenbestimmung einzelner DNA-Sequenzen. Sie ist eine Variante der Polymerase-Kettenreaktion.
, Digital polymerase chain reaction (digital … Digital polymerase chain reaction (digital PCR, DigitalPCR, dPCR, or dePCR) is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample ction within a sample, however the sample
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, Digital polymerase chain reaction
, Digital polymeraskedjereaktion
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