ddPCR原理与应用简介
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Experimental design: This assay exploits a correlation, reported herein, between the length of an amplified DNA molecule and the fluorescence amplitude produced in droplet digital PCR (ddPCR), to allow the user to calculate the size of unknown DNA.
Conclusion: The frequency distribution shows a high degree of overlap and
a common center point for the estimation made using the QuantiSize assay and the observations from the MiSeq.
稀有突变碱基或稀有序列的检测
Results: All 9 patients exhibited a plasma response to erlotinib,
with 8 demonstrating a complete plasma response. In 6 of the patients, plasma levels of mutant EGFR
Conclusion: digital PCR of plasma DNArmination of HER2 status. This approach could be adapted to the assessment of any amplified locus in cancer and in particular may be a useful strategy screening for potentially rare acquisition events in response to therapy.
Results: Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR.
ddPCR与NGS
Purpose: We have developed a new assay “QuantiSize” capable of concurrently measuring the absolute concentration and the length of unknown amplifiable DNA templates, making it well suited.
达、CNV分析等
Purpose&Experimental Design： In this study, we constructed a plasmid containing the amoA gene of AOA (A-amoA) and AOB (B-amoA), and the nosZ and nirS genes of denitrifiers. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material.
Droplet digital PCR(ddPCR)微滴式数字PCR 原理与应用简介
工作原理
技术优势
医学应用
Part 1
工作原理
PCR发展历程
• 普通PCR • 定性分析
第一代
第二代
• Real-time PCR • 间接定量分析 • 依赖Ct或Cq值，标准品
• 数字PCR • 直接定量分析 • 无需标准品 • 绝对定量
Results: The results were consistent with the results presented above, ddPCR showed greater precision.
Conclusion: Nanoliter-sized droplet technology paired with digital PCR(ddPCR) holds promise for highly precise, absolute nucleic acid quantification.
were again detected at objective progression, with plasma progression detected 4-12 weeks prior to RECIST progression. The remaining 3 patients had no reemergence of plasma genotype at objective progression.
The QuantiSize assay has the potential to increase the average yield of usable data generated from sequencing runs, thereby increasing the efficiency and throughput.
Conclusion: Droplet digital PCR is an attractive technology as its speed, cost, and ease of use is similar to other PCR-based assays, yet the sensitivity and quantitative nature of this assay offers broader clinical application.
第三代
反应体系配制
样品、引物和探针 /EvaGreen染料与预混 液混合
微滴制作
将反应体系加入微滴 发生卡中 目的片段和背景序列 随机分布到微滴内
微滴PCR扩增
将微滴化处理的样品 转移至96孔PCR反应板 中，并封膜，进行PCR 扩增
读取分析结果
将反应板置于微滴分 析仪内，对微滴荧光 信号逐个检测、分析
Conclusion: This method has the potential to accurately quantify DNA reference materials.
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对样品中的DNA进行绝对定量
达、CNV分析等
微滴化实现了稀有序列或稀有突变碱基的富集
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对动样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
达、CNV分析等
Results: The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC – IDMS, improving both the accuracy and reliability of the plasmid reference material.
Part 3
医学应用
稀有突变碱基或稀有序列的检测
Purpose: To determine whether ddPCR could identify the development of resistance mutations after treatment with targeted therapy, we studied patients with advanced EGFR-mutant NSCLC treated on a prospective clinical trial of first-line erlotinib (NCT00997334).
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
TaqMan探针
EvaGreen染料
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
拷贝数变异检测
Purpose: Compare biopsy DNA tested with FISH to plasma DNA tested with droplet digital PCR to identify known HER2 amplified and non-amplified samples.
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
达、CNV分析等
Purpose&Experimental Design： We describe a series of experiments to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR (Bio-Rad Laboratories, Hercules, CA, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA and heparin) directly into the PCR reactions
Conclusion: ddPCR may offer an advantage over qPCR when dealing with inhibition prone specimens. ddPCR may prove especially useful for clinical sample types such as stool, sputum, and tissue are known to be more recalcitrant to removal of inhibitors through typical extraction methods.
Results: ddPCR was used to detect HER2 copy number amplifications in both cell-lines and circulating tumor DNA from plasma. All samples had previously been tested using FISH.
Part 2
技术优势
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对动样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
Conclusion: The frequency distribution shows a high degree of overlap and
a common center point for the estimation made using the QuantiSize assay and the observations from the MiSeq.
稀有突变碱基或稀有序列的检测
Results: All 9 patients exhibited a plasma response to erlotinib,
with 8 demonstrating a complete plasma response. In 6 of the patients, plasma levels of mutant EGFR
Conclusion: digital PCR of plasma DNArmination of HER2 status. This approach could be adapted to the assessment of any amplified locus in cancer and in particular may be a useful strategy screening for potentially rare acquisition events in response to therapy.
Results: Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR.
ddPCR与NGS
Purpose: We have developed a new assay “QuantiSize” capable of concurrently measuring the absolute concentration and the length of unknown amplifiable DNA templates, making it well suited.
达、CNV分析等
Purpose&Experimental Design： In this study, we constructed a plasmid containing the amoA gene of AOA (A-amoA) and AOB (B-amoA), and the nosZ and nirS genes of denitrifiers. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material.
Droplet digital PCR(ddPCR)微滴式数字PCR 原理与应用简介
工作原理
技术优势
医学应用
Part 1
工作原理
PCR发展历程
• 普通PCR • 定性分析
第一代
第二代
• Real-time PCR • 间接定量分析 • 依赖Ct或Cq值，标准品
• 数字PCR • 直接定量分析 • 无需标准品 • 绝对定量
Results: The results were consistent with the results presented above, ddPCR showed greater precision.
Conclusion: Nanoliter-sized droplet technology paired with digital PCR(ddPCR) holds promise for highly precise, absolute nucleic acid quantification.
were again detected at objective progression, with plasma progression detected 4-12 weeks prior to RECIST progression. The remaining 3 patients had no reemergence of plasma genotype at objective progression.
The QuantiSize assay has the potential to increase the average yield of usable data generated from sequencing runs, thereby increasing the efficiency and throughput.
Conclusion: Droplet digital PCR is an attractive technology as its speed, cost, and ease of use is similar to other PCR-based assays, yet the sensitivity and quantitative nature of this assay offers broader clinical application.
第三代
反应体系配制
样品、引物和探针 /EvaGreen染料与预混 液混合
微滴制作
将反应体系加入微滴 发生卡中 目的片段和背景序列 随机分布到微滴内
微滴PCR扩增
将微滴化处理的样品 转移至96孔PCR反应板 中，并封膜，进行PCR 扩增
读取分析结果
将反应板置于微滴分 析仪内，对微滴荧光 信号逐个检测、分析
Conclusion: This method has the potential to accurately quantify DNA reference materials.
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对样品中的DNA进行绝对定量
达、CNV分析等
微滴化实现了稀有序列或稀有突变碱基的富集
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对动样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
达、CNV分析等
Results: The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC – IDMS, improving both the accuracy and reliability of the plasmid reference material.
Part 3
医学应用
稀有突变碱基或稀有序列的检测
Purpose: To determine whether ddPCR could identify the development of resistance mutations after treatment with targeted therapy, we studied patients with advanced EGFR-mutant NSCLC treated on a prospective clinical trial of first-line erlotinib (NCT00997334).
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
TaqMan探针
EvaGreen染料
ddPCR的工作流程：QX200 ddPCR
ddPCR的工作流程：QX200 ddPCR
拷贝数变异检测
Purpose: Compare biopsy DNA tested with FISH to plasma DNA tested with droplet digital PCR to identify known HER2 amplified and non-amplified samples.
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表
达、CNV分析等
Purpose&Experimental Design： We describe a series of experiments to compare the inhibition tolerance of laboratory-developed CMV qPCR and ddPCR (Bio-Rad Laboratories, Hercules, CA, QX-100) assays by introducing a panel of clinically relevant inhibitors (SDS, EDTA and heparin) directly into the PCR reactions
Conclusion: ddPCR may offer an advantage over qPCR when dealing with inhibition prone specimens. ddPCR may prove especially useful for clinical sample types such as stool, sputum, and tissue are known to be more recalcitrant to removal of inhibitors through typical extraction methods.
Results: ddPCR was used to detect HER2 copy number amplifications in both cell-lines and circulating tumor DNA from plasma. All samples had previously been tested using FISH.
Part 2
技术优势
01 实现稀有碱基或稀有序列的高灵敏检测，检测限低至0.001%
ddPCR 技术优势
02 无需标准品，即可对动样品中的DNA进行绝对定量
03
终点PCR检测，不依赖Ct值或扩增效率，能克服PCR抑制 剂的影响，因而对样本的复杂性容忍度较高
04 准确度、精密度和重复性较高，可精确测定靶基因的相对表