کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2034721 1543051 2016 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms
ترجمه فارسی عنوان
بهینه سازی واکنش زنجیره ای پلی مراز قطره دیجیتال برای اندازه گیری ارگانیسم های اصلاح شده ژنتیکی
موضوعات مرتبط
علوم زیستی و بیوفناوری علوم کشاورزی و بیولوژیک علوم کشاورزی و بیولوژیک (عمومی)
چکیده انگلیسی


• Experience matrix condenses ddPCR performance parameters in graphical presentation.
• Assay separation value based on absolute fluorescence signal distance and variation.
• Separation value and experience matrix simplify choice of best assay parameters.
• Influence of oligonucleotide concentration and annealing/extension temperature.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called ‘rain’. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis.We developed an Excel based ‘experience matrix’ that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations.The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

Figure optionsDownload as PowerPoint slide

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biomolecular Detection and Quantification - Volume 7, March 2016, Pages 9–20
نویسندگان
, , , ,