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PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

Identifikátory výsledku

  • Kód výsledku v IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216305%3A26620%2F20%3APU137798" target="_blank" >RIV/00216305:26620/20:PU137798 - isvavai.cz</a>

  • Výsledek na webu

    <a href="https://pubs.acs.org/doi/pdf/10.1021/acsomega.0c04766" target="_blank" >https://pubs.acs.org/doi/pdf/10.1021/acsomega.0c04766</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.1021/acsomega.0c04766" target="_blank" >10.1021/acsomega.0c04766</a>

Alternativní jazyky

  • Jazyk výsledku

    angličtina

  • Název v původním jazyce

    PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

  • Popis výsledku v původním jazyce

    Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K· s−1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

  • Název v anglickém jazyce

    PCR Multiplexing Based on a Single Fluorescent Channel Using Dynamic Melting Curve Analysis

  • Popis výsledku anglicky

    Since its invention in 1986, the polymerase chain reaction (PCR), has become a well-established method for the detection and amplification of deoxyribonucleic acid (DNA) with a specific sequence. Incorporating fluorescent probes, known as TaqMan probes, or DNA intercalating dyes, such as SYBR Green, into the PCR mixture allows real-time monitoring of the reaction progress and extraction of quantitative information. Previously reported real-time PCR product detection using intercalating dyes required melting curve analysis (MCA) to be performed following thermal cycling. Here, we propose a technique to perform dynamic MCA during each thermal cycle, based on a continuous fluorescence monitoring method, providing qualitative and quantitative sample information. We applied the proposed method in multiplexing detection of hepatitis B virus DNA and complementary DNA of human immunodeficiency virus as well as glyceraldehyde 3-phosphate dehydrogenase in different concentration ratios. We extracted the DNA melting curve and its derivative from each PCR cycle during the transition from the elongation to the denaturation temperature with a set heating rate of 0.8 K· s−1and then used the data to construct individual PCR amplification curves for each gene to determine the initial concentration of DNA in the sample. Our proposed method allows researchers to look inside the PCR in each thermal cycle, determining the PCR product specificity in real time instead of waiting until the end of the PCR. Additionally, the slow transition rate from elongation to denaturation provides a dynamic multiplexing assay, allowing the detection of at least three genes in real time.

Klasifikace

  • Druh

    J<sub>imp</sub> - Článek v periodiku v databázi Web of Science

  • CEP obor

  • OECD FORD obor

    10406 - Analytical chemistry

Návaznosti výsledku

  • Projekt

    Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

  • Návaznosti

    P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Ostatní

  • Rok uplatnění

    2020

  • Kód důvěrnosti údajů

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Údaje specifické pro druh výsledku

  • Název periodika

    ACS OMEGA

  • ISSN

    2470-1343

  • e-ISSN

  • Svazek periodika

    5

  • Číslo periodika v rámci svazku

    46

  • Stát vydavatele periodika

    US - Spojené státy americké

  • Počet stran výsledku

    7

  • Strana od-do

    30267-30273

  • Kód UT WoS článku

    000595527600064

  • EID výsledku v databázi Scopus