http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–5 ! 2014 Informa UK Ltd. DOI: 10.3109/19401736.2014.913168
SHORT COMMUNICATION
Droplet digital PCR technology promises new applications and research areas P. Manoj
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 10/06/14 For personal use only.
Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala, India
Abstract
Keywords
Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR.
Gene copy number, qPCR, real-time PCR
Introduction Real-time PCR is commonly used in disease related research, in clinical diagnosis, in disease progression detection and in preventive medicine due to its speed, sensitivity, accuracy, and ease of use. In real-time quantitative PCR (qPCR), DNA is amplified until it produces a certain level of signal. The level at which the reaction fluorescence crosses an intensity threshold is called the cycle threshold. The number of cycles needed to reach this point is then used to calculate the amount of DNA molecules originally present in the sample. Real-time PCR can be used to do: (a) absolute quantification of nucleic acids with or without a standard curve, (b) relative quantification by comparing with a reference sample and endogenous control using cycle threshold (CT) value. But the CT value calculation may go wrong on account of many factors like, initial template concentration, absence of the correct endogenous control and the standardization of the procedure. Digital PCR concept was conceived in 1992 using nested PCR. The basis of digital PCR (dPCR) is to quantify the absolute number of target present in a sample, using limiting dilutions, PCR and poisson statistics (Sykes et al., 1992). It has been shown to be a promising tool for cancer genomic content determination, including sequencing human genome (Vogelstein & Kinzler, 1999). A bead, emulsion, amplification, magnetics
Correspondence: Dr. P. Manoj, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala, 695014, India. Tel: +91 4712529438, +91 09249939990. Fax: +91 4712348096. E-mail: [email protected]
History Received 21 January 2014 Revised 2 April 2014 Accepted 5 April 2014 Published online 29 April 2014
technology (BEAMing), using emulsion beads for digital PCR, was done by Dressman (Dressman et al., 2003) and dPCR proved useful for the analysis of heterogenous methylation (Mikeska et al., 2010). dPCR uses the same primers and probes as qPCR but is capable of higher sensitivity and precision. In qPCR gene expression differences or copy number variants smaller than 2-fold cannot be distinguished. Identifying alleles with frequencies of less than about 1% is difficult because it would also detect highly abundant common alleles with similar sequences. dPCR can measure smaller differences in gene-expression and can identify alleles occurring at a frequency of one in thousands. Today, digital PCR is poised to make a significant impact in the diagnostics field by the detection of nucleic acids at higher resolution and lower target levels. It does not require the calibration and internal controls necessary for qPCR. Digital PCR has the ability to identify diseases earlier in progression, providing an advantage for diagnostics and preventive medicine. dPCR able to track the presence, expansion and disappearance of pathogenic organisms, genetic variants in cancer, infectious diseases and other human diseases. The present ddPCR systems produce hundreds to millions of minute partitions of the reactions. Its ability to precisely analyze nucleic acids at a molecular level makes it ideal for tasks such as detecting copy number variation and point mutations. Droplet digital PCR (ddPCR) is an upgradation of real-time PCR that enable us to directly quantify and clonally amplify nucleic acids (DNA and RNA), which provides an absolute measure of target DNA molecule and delivers an absolute count of the number of target sequences (Table 1).
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P. Manoj
Mitochondrial DNA, Early Online: 1–5
Table 1. Real time qPCR versus digital PCR. Real Time qPCR Measures PCR amplification as it occurs. Data is collected during the log phase of PCR
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Applications Quantitation of Gene Expression Microarray Verification Pathogen detection/Viral Quantitation SNP Genotyping, Copy Number Variation MicroRNA Analysis siRNA/RNAi experiments Advantages Increased dynamic range of detection Detection is capable down to a 2-fold change Collects data in the exponential growth phase of PCR An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
The ddPCR system is used to find absolute and relative quantification of targets, without the need for calibration curves. ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors (Whale et al., 2012). It is identified as a new tool to accurately analyze extremely rare mitochondrial DNA (mtDNA) deletions associated with a range of diseases and disorders as well as aging (Taylor et al., 2014).
Different platforms of dPCR Digital PCR gives a high impact on the research applications. Out of 2 major platforms, one is droplet technology where microfluidics is used to emulsify samples in oil to create droplets. This lead to one DNA per droplet and each droplet is subject to PCR many times. Raindance technologies and Bio-Rad laboratories are using this technology by which the former produces picolitre droplets (raindance) and the later creates nanolitre droplets (Biorad, Hercules, CA). In 2006 the 1st digital PCR based on integrated fluidic circuits (chips), with chambers and valves was introduced by Fluidigm (Pekin et al., 2011) and BAEMing dPCR sevices by Inostics began in 2008 (Jian et al., 2008). Fluidigm and Life Tech (Quantstudio) create reaction chambers within specially designed chips or plates. Here also one DNA per partition is created, the PCR amplification and fluorescent detection is done. In 2010, Life Technologies commercialized a digital PCR for the open array system. Quantalife developed the droplet digital PCR (ddPCR) technology, in which a sample is partitioned into 20,000 droplets and provide a digital counting of nucleic acid targets (Andrew et al., 2011). In 2011, Quantalife was acquired by BioRad Laboratories and in 2012. In the chip-based platforms upto 20,000 partitions are made at once, and the advantage is that each partition holds a fixed volume (Pinheiro et al., 2012; Sanders et al., 2011). Digital PCR (dPCR) is an end-point measurement that helps to quantify nucleic acids without the use of standard curves. In ddPCR, a sample is diluted and partitioned into hundreds or even millions of separate reaction chambers, so that each chamber contains one or no copies of the sequence of interest (Chen et al., 2012). In droplet digital PCR, reaction chambers are separated by carefully titrated emulsion of oil, water and stabilizing chemicals. The samples are dispersed into tiny droplets, and the droplets of each sample are transferred into plates for thermo cycling. After PCR the plates are transferred to a droplet reading machine which functions like a flow cytometer which analyzes each droplet and
Digital PCR Partitioning a sample into many individual reactions; some reactions contain the target molecule (positive) while others do not (negative) from which generate the exact number of target molecules in the sample, without reference to standards or endogenous controls Applications Absolute quantification of viral load Absolute quantification of nucleic acid Absolute quantification of NGS libraries Rare event detection Copy number variation Enrichment and separation of mixtures Advantages No need to rely on references or standards, desired precision by increasing total number of PCR replicates Highly tolerant to inhibitors Capable of analyzing complex mixtures Provides a linear response to the number of copies present to allow for small fold change differences to be detected
checks whether a reaction has occurred or not. By counting the number of positive partitions and negative partitions, one can determine exactly how many copies of a DNA molecule were there in the original sample. It is used to distinguish differential expression of a variety of alleles (Chen et al., 2012), to track which viruses infect individual bacterial cells (Tadmoret et al., 2011), to quantify cancer genes in patient specimens (Wang et al., 2010) and to detect copy number variation in breast cancer cells (Lo et al., 2007).
QX100, Quant Studio, Raindrop & BiomarkHD The QX100 Droplet Digital PCR system from Bio-Rad Laboratories Inc. is a newer addition to the ddPCR instruments along with Quant Studio 12 K Flex system of Life technologies and Raindrop digital PCR of Rain Dance. Common KRAS mutations were screened in 2 multiplex experiments using Rain Dance dPCR and could detect mutant KRAS gene within 200,000 wild type KRAS genes (Pekin et al., 2011). Using BioMark HD of Fluidigm, small difference in copy number could be identified which was approximately between six and seven copies of the specific gene (Jian et al., 2008). Wide-field fluorescence imaging by Digital PCR was used for analysis of single cells, organisms or molecules (Andrew et al., 2011).
Droplet digital PCR (ddPCR) ddPCR is used in phage-host interaction study (Tadmoret et al., 2011), fetal screening (Lo et al., 2007), biomarker analysis (Day et al., 2013), viral detection(Shen et al., 2011), prognostic monitoring (Belgrader et al., 2013), intracellular profiling (Sindelka et al., 2008), detection of GMO in food (Morisset et al., 2013), in high throughput sequencing (White et al., 2009), mitochondrial DNA alteration in Alzheimer disease (Podlesniy et al., 2013), and in mitochondrial DNA mutations (Taylor et al., 2014). QX100 Droplet Digital PCR system partitions the nucleic acid sample into approximately 20,000 droplets, with target and background DNA randomly distributed among them. After PCR amplification occurs, a reader determines which droplets contain a target (positive) and which do not (negative). ddPCR quantifies DNA copy number by partitioning PCR reactions into droplets (i.e. 20,000 separate reactions each containing a fluorescentlabeled probe) and then counting positive droplets, enabling enumeration of amplifiable molecules in the starting sample. The readout is the number of PCR plus and PCR minus droplets
Droplet digital PCR technology
DOI: 10.3109/19401736.2014.913168
standard curve and the concentration calculation is done based on Poisson algorithm which claims more accuracy and reproducibility (Lin, 2012).
Methodology Following standard protocol using TRIZOL reagent, 20 ng cDNA prepared. The ddPCR work flow starts by partitioning the TaqMan reaction mix containing cDNA into droplets in oil using droplet generator. PCR reaction mixtures of 20 mL volume comprising 1 ddPCR Master Mix (Bio-Rad-186-3010), relevant primer and probe mix (20) (900 nM each primer, 250 nM probe)
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from each reaction. The software calculates the concentration of target DNA as copies per microliter. The system uses microfluidic circuits and surfactant chemistries to divide a microliter mixture of sample and reagents into the monodisperse droplets, which support PCR amplification of single template molecules using homogeneous assay chemistries and workflows similar to those used for real-time PCR applications. An automated droplet flow cytometer reads each set of droplets after PCR at a rate of 32 wells per hour (Baker, 2012). Digital PCR has more applications in cancer research, mutation detection, prenatal diagnosis and copy number variation studies. The method uses no CT value, endogenous control or
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Figure 1. (A) Scatter plot showing positive and negative partions. (B) Histogram showing positive and negative partions. (C) Plot showing concentration (copies/microliter) based on poisson algorithm. (D) Plot showing events vs sample, demarking positive, negative and accepted droplets.
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P. Manoj
and DNA were prepared. Each 20 mL reaction was dispensed into a separate well of a disposable eight channel droplet generator cartridge (Bio-Rad DG8 catridges and gaskets, 186–3006). No Template Controls (NTC) containing 1 TE0.1 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) in place of DNA were also used in the reaction set up. 70 mL of droplet generation oil (186–3005) was then added into each of the corresponding oil wells before the chip was loaded into the droplet generator (QX100, Bio-Rad). The droplet generator applies a vacuum to each of the outlet wells to generate droplets in the eight channels simultaneously at a rate of 1000 droplets per channel per second until the complete 20 mL ddPCR mixture has been partitioned into the water-in-oil emulsion format. Each water-in-oil emulsion (40 mL) was transferred using multichannel pipette to separate wells in a 96well polypropylene plate (Eppendorf), sealed and amplified in a conventional thermal cycler (C1000 Touch Thermocycler-Bio rad). Two step thermo cycling protocol consisted of a 10 min activation period at 95 C followed by 40 cycles of 30 s at 94 C denaturation and 60 s at 60 C for combined annealing-extension at ramp rate set to 2.5 C/sec) ramp rate and a final 10 min inactivation step at 98 C. After thermal cycling, plates were heat sealed with aluminium sealer, then transferred to QX 100 droplet reader (Bio-Rad) containing droplet reader oil (186–3004), that employs an integrated auto sampler and fluidics to serially aspirate droplets from each well and stream them single-file, at a rate of 1500 droplets/second, past a two-color fluorescence detector sampled at a rate of 100 kHz on both FAM and VIC fluorescence channels. Discrimination between droplets that did not contain target (negatives) and those which did (positives) was achieved by applying a global fluorescence amplitude threshold in Quanta Soft (Bio-Rad), the software package provided with the ddPCR system for data acquisition and analysis. The droplet reader was calibrated once upon installation of the ddPCR system at the Institute RGCB, India. The simple calibration procedure for generation of an instrument-specific color compensation matrix was done and that was stored on the droplet reader and automatically applied to data to eliminate cross talk between FAM and VIC labeled probes. Quanta Soft uses a proprietary signal-processing algorithm to automatically perform droplet gating within each run. The threshold was set as the midpoint between the average fluorescence amplitude of positives and negative droplet clusters on each of the FAM and VIC channels.
Results and discussion ‘‘Quanta soft’’ application software (Bio-rad) was used for data analysis with default settings. A series of experiments were designed to detect dioeciousness of Piper longum plants for breeding purpose. cDNA was made from purified RNA of fresh leaf samples of male and female plants of piper, quantified, diluted, and used for ddPCR. Three replicates of each diluted DNA were prepared in eight-well plates using droplet generator cartridges. Calculation of copy number was based on Poisson distribution. The scatter diagram, and histogram, showed the positive and negative droplets of the target gene, from which copy number per microliter of sample was calculated by poisson equation. Events, number of positive droplets, negative droplets and accepted droplets were shown in graph (Figure 1). Multiplex reaction was also performed using FAM and VIC labeled probes for our control material with 2 target gene. Manual pippetting of template and primer/probes resulted in varying amplitude of positive droplets causing different signal to noise ratios and difficulty in setting a threshold for positive/negative droplets. NTC has shown low-level background indicating very less
Mitochondrial DNA, Early Online: 1–5
contamination during sample handling. Based on the number of positive partitions, the concentration was calculated using the formula: M ¼ ln [1[P/R]] copy number per droplet [9]. This calculation is based on the assumption that all the partitions are of equal volume. Poisson statistics are needed to account a positive PCR partition containing more than one molecule. The amount of template used for the experiment is an important factor in dPCR. Absorbance measurements of the DNA are taken at 260 nm. Concentration is calculated by dividing the copy number by the reaction volume, in which the size of the droplet is also to be considered for accuracy provided the droplets remain a uniform size even when the temperature fluctuates slightly. An accurate estimation of partition volume is important because different instruments may have different partition volumes. The sum of the partitions multiplied by partition volume will give the total volume of the reaction. DNA copy number concentration was around 1000 to 17,000. On binomial approximation 99.5% saturation of the droplets means 10 negative droplets in a ddPCR containing 20,000 droplets indicate between 10,200 and 110,000 copies of target DNA. Here the report is on a pilot study using plant specific primers against cDNA from different plants. Here we have used well identified male and female plant samples for the study and the experiment termed to be successful in identifying the sex of the plant samples, where FAM labelled primers shown amplification only from female plants and VIC labelled primers in male plants only. In this study only six primers were used and the experiment to be continued with more number of primers to generate sufficient data.
Conclusion Droplet Digital PCR has become an emerging tool for new applications and research. It is used in quantification of miRNAs, pathogens, in rare allele detection, and copy number variation detection. The accumulation of mt DNA mutations associated with aging, neuromuscular disorders and cancer are quantified through droplet digital PCR. The method overrides the variation of amplification, with high level of reliability and reproducibility. Since it is carried out in minute volumes and in single molecule concentrations, it has to be done with accuracy. It is an end point assay, and after PCR each partition will contain a negative or positive target of the nucleic acids which will be quantified by counting positive and negative reactions. As in qPCR inhibitors can also create false negatives by blocking the reaction to occur. Prior to the assay designing we should make sure that there is only one target. Multiple targets can be separated as different partitions in different wells. In case of rare allele detection, rates of false positives are very crucial. It may mask the number of true positives. Getting an estimate of the initial concentration of the template may help in making appropriate dilution of the sample. Droplet Digital PCR technology is highly reproducible with higher specificity sensitivity and accuracy, which can detect targets among fragments of DNA and has a unique ability to perform sensitive molecular measurements without the need of a standard curve. The conversion of real-time PCR analogue signal to binary output simplifies the measurement considerably. The major strength of ddPCR is the direct counting approach which provides precise, quantitative measurements without reference to a calibrator.
Declaration of interest The author declares no competing interests by conducting this study.
DOI: 10.3109/19401736.2014.913168
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References Andrew CH, Jeffrey SF, Armando RT, Albert TH, Robert L, Stephen LP, David LY, Abraham PL. (2011). 1-million droplet array with wide-field fluorescence imaging for digital PCR. Lab Chip 11:3838–45. Baker M. Digital PCR hits its stride. Nat Methods 6:541–6. Belgrader P, Tanner SC, Regan JF, Koehler R, Hindson BJ, Brown AS. (2013). Droplet digital PCR measurement of HER2 copy number alterations in formalin fixed paraffin-embedded breast carcinoma tissue. Clin Chem 59:991–4. Chen R, Mias GI, Li-Pook-Than J, Jiang L, Lam HYK, Chen R, Miriami E, et al. (2012). Personal omics profiling reveals dynamic molecular and medical phenotypes. Cell 148:1293–307. Day E, Dear PH, McCaughan F. (2013). Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine. Methods 59:101–7. Dennis Lo YM, Lun FMF, Allen Chen KC, Tsui NBY, Chong KC, Lau LT, Leung TY, et al. (2007). Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc Natl Acad Sci USA 104:13116–21. Dressman D, Yan H, Traverso G, Kinzler K.W, Vogelstein B. (2003). Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc Natl Acad Sci USA 100:8817–22. Jian Q, Jones RC, Ramakrishnan R. (2008). Studying copy number variations using a nanofluidic platform. Nucleic Acids Res 36:e116. doi: 10.1093/nar/gkn518. Lin CYB. (2012). Recent advances in digital PCR. Prog Chem 24: 2415–23. Mikeska T, Candiloro IL, Dobrovic A. (2010). The implications of heterogenous DNA methylation for the accurate quantification of methylation. Epigenomics 2:561–73. Morisset D, Stebih D, Milavec M, Gruden K, Zel J. (2013). Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One 8:e62583. Pekin D, Skhiri Y, Baret JC, LeCorre D, Mazutis L, Salem CB, Millot F, et al. (2011). Quantitative and sensitive detection of rare mutations using droplet-based microfluidics. Lab Chip 11:2156–66. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR, et al. (2012). Evaluation of a droplet digital
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polymerase chain reaction format for DNA copy number quantification. Anal Chem 84:1003–11. Podlesniy P, Figueiro-Silva J, Llado A, Antonell A, Scanchez-valle R, Alcolea D, Lleo A, et al. (2013). Low cerebrospinal fluid concentration of mtDNA in preclinical Alzheimer disease. Ann Neurobiol 74: 655–68. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. (2011). Evaluation of digital PCR for absolute DNA quantification. Anal Chem 83:6474–84. Shen F, Sun B, Kreutz JE, Davydova EK, Du W, Reddy PL, Joseph LJ, Ismagilov RF. (2011). Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational slip-chip tested with HIV and hepatitis C viral load. J Am Chem Soc 133:17705–12. Sindelka R, Jonak J, Hands R, Bustin SA, Kubista M. (2008). Intercellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte. Nucleic Acids Res 36:387–92. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA. (1992). Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13:444–9. Tadmor AD, Ottesen EA, Leadbetter JR, Phillips R. Probing individual environmental bacteria for viruses by using microfluidic digital PCR. Science 333:58–62. Taylor SD, Ericson NG, Burton JN, Prolla TA, Silber JR, Shendure J, Bielas JH. (2014). Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain. Aging Cell 13:29–38. Vogelstein B, Kinzler KW. (1999). Digital PCR. Proc Natl Acad Sci USA 96:9236–41. Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, Foy CA, Scott DJ. (2012). Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res 40:e82. Wang JR, Ramakrishnan R, Tang Z, Fan W, Kluge A, Dowlati A, Jones RC, Ma PC. (2010). Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays. Clin Chem 56:623–32. White RA, Blainey PC, Fan HC, Quake SR. (2009). Digital PCR provides sensitive and absolute calibration for high throughput sequencing. BMC Genomics 10:116.
SHORT COMMUNICATION
Droplet digital PCR technology promises new applications and research areas P. Manoj
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 10/06/14 For personal use only.
Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala, India
Abstract
Keywords
Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR.
Gene copy number, qPCR, real-time PCR
Introduction Real-time PCR is commonly used in disease related research, in clinical diagnosis, in disease progression detection and in preventive medicine due to its speed, sensitivity, accuracy, and ease of use. In real-time quantitative PCR (qPCR), DNA is amplified until it produces a certain level of signal. The level at which the reaction fluorescence crosses an intensity threshold is called the cycle threshold. The number of cycles needed to reach this point is then used to calculate the amount of DNA molecules originally present in the sample. Real-time PCR can be used to do: (a) absolute quantification of nucleic acids with or without a standard curve, (b) relative quantification by comparing with a reference sample and endogenous control using cycle threshold (CT) value. But the CT value calculation may go wrong on account of many factors like, initial template concentration, absence of the correct endogenous control and the standardization of the procedure. Digital PCR concept was conceived in 1992 using nested PCR. The basis of digital PCR (dPCR) is to quantify the absolute number of target present in a sample, using limiting dilutions, PCR and poisson statistics (Sykes et al., 1992). It has been shown to be a promising tool for cancer genomic content determination, including sequencing human genome (Vogelstein & Kinzler, 1999). A bead, emulsion, amplification, magnetics
Correspondence: Dr. P. Manoj, Rajiv Gandhi Centre for Biotechnology, Thycaud P.O., Thiruvananthapuram, Kerala, 695014, India. Tel: +91 4712529438, +91 09249939990. Fax: +91 4712348096. E-mail: [email protected]
History Received 21 January 2014 Revised 2 April 2014 Accepted 5 April 2014 Published online 29 April 2014
technology (BEAMing), using emulsion beads for digital PCR, was done by Dressman (Dressman et al., 2003) and dPCR proved useful for the analysis of heterogenous methylation (Mikeska et al., 2010). dPCR uses the same primers and probes as qPCR but is capable of higher sensitivity and precision. In qPCR gene expression differences or copy number variants smaller than 2-fold cannot be distinguished. Identifying alleles with frequencies of less than about 1% is difficult because it would also detect highly abundant common alleles with similar sequences. dPCR can measure smaller differences in gene-expression and can identify alleles occurring at a frequency of one in thousands. Today, digital PCR is poised to make a significant impact in the diagnostics field by the detection of nucleic acids at higher resolution and lower target levels. It does not require the calibration and internal controls necessary for qPCR. Digital PCR has the ability to identify diseases earlier in progression, providing an advantage for diagnostics and preventive medicine. dPCR able to track the presence, expansion and disappearance of pathogenic organisms, genetic variants in cancer, infectious diseases and other human diseases. The present ddPCR systems produce hundreds to millions of minute partitions of the reactions. Its ability to precisely analyze nucleic acids at a molecular level makes it ideal for tasks such as detecting copy number variation and point mutations. Droplet digital PCR (ddPCR) is an upgradation of real-time PCR that enable us to directly quantify and clonally amplify nucleic acids (DNA and RNA), which provides an absolute measure of target DNA molecule and delivers an absolute count of the number of target sequences (Table 1).
2
P. Manoj
Mitochondrial DNA, Early Online: 1–5
Table 1. Real time qPCR versus digital PCR. Real Time qPCR Measures PCR amplification as it occurs. Data is collected during the log phase of PCR
Mitochondrial DNA Downloaded from informahealthcare.com by University of Otago on 10/06/14 For personal use only.
Applications Quantitation of Gene Expression Microarray Verification Pathogen detection/Viral Quantitation SNP Genotyping, Copy Number Variation MicroRNA Analysis siRNA/RNAi experiments Advantages Increased dynamic range of detection Detection is capable down to a 2-fold change Collects data in the exponential growth phase of PCR An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
The ddPCR system is used to find absolute and relative quantification of targets, without the need for calibration curves. ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors (Whale et al., 2012). It is identified as a new tool to accurately analyze extremely rare mitochondrial DNA (mtDNA) deletions associated with a range of diseases and disorders as well as aging (Taylor et al., 2014).
Different platforms of dPCR Digital PCR gives a high impact on the research applications. Out of 2 major platforms, one is droplet technology where microfluidics is used to emulsify samples in oil to create droplets. This lead to one DNA per droplet and each droplet is subject to PCR many times. Raindance technologies and Bio-Rad laboratories are using this technology by which the former produces picolitre droplets (raindance) and the later creates nanolitre droplets (Biorad, Hercules, CA). In 2006 the 1st digital PCR based on integrated fluidic circuits (chips), with chambers and valves was introduced by Fluidigm (Pekin et al., 2011) and BAEMing dPCR sevices by Inostics began in 2008 (Jian et al., 2008). Fluidigm and Life Tech (Quantstudio) create reaction chambers within specially designed chips or plates. Here also one DNA per partition is created, the PCR amplification and fluorescent detection is done. In 2010, Life Technologies commercialized a digital PCR for the open array system. Quantalife developed the droplet digital PCR (ddPCR) technology, in which a sample is partitioned into 20,000 droplets and provide a digital counting of nucleic acid targets (Andrew et al., 2011). In 2011, Quantalife was acquired by BioRad Laboratories and in 2012. In the chip-based platforms upto 20,000 partitions are made at once, and the advantage is that each partition holds a fixed volume (Pinheiro et al., 2012; Sanders et al., 2011). Digital PCR (dPCR) is an end-point measurement that helps to quantify nucleic acids without the use of standard curves. In ddPCR, a sample is diluted and partitioned into hundreds or even millions of separate reaction chambers, so that each chamber contains one or no copies of the sequence of interest (Chen et al., 2012). In droplet digital PCR, reaction chambers are separated by carefully titrated emulsion of oil, water and stabilizing chemicals. The samples are dispersed into tiny droplets, and the droplets of each sample are transferred into plates for thermo cycling. After PCR the plates are transferred to a droplet reading machine which functions like a flow cytometer which analyzes each droplet and
Digital PCR Partitioning a sample into many individual reactions; some reactions contain the target molecule (positive) while others do not (negative) from which generate the exact number of target molecules in the sample, without reference to standards or endogenous controls Applications Absolute quantification of viral load Absolute quantification of nucleic acid Absolute quantification of NGS libraries Rare event detection Copy number variation Enrichment and separation of mixtures Advantages No need to rely on references or standards, desired precision by increasing total number of PCR replicates Highly tolerant to inhibitors Capable of analyzing complex mixtures Provides a linear response to the number of copies present to allow for small fold change differences to be detected
checks whether a reaction has occurred or not. By counting the number of positive partitions and negative partitions, one can determine exactly how many copies of a DNA molecule were there in the original sample. It is used to distinguish differential expression of a variety of alleles (Chen et al., 2012), to track which viruses infect individual bacterial cells (Tadmoret et al., 2011), to quantify cancer genes in patient specimens (Wang et al., 2010) and to detect copy number variation in breast cancer cells (Lo et al., 2007).
QX100, Quant Studio, Raindrop & BiomarkHD The QX100 Droplet Digital PCR system from Bio-Rad Laboratories Inc. is a newer addition to the ddPCR instruments along with Quant Studio 12 K Flex system of Life technologies and Raindrop digital PCR of Rain Dance. Common KRAS mutations were screened in 2 multiplex experiments using Rain Dance dPCR and could detect mutant KRAS gene within 200,000 wild type KRAS genes (Pekin et al., 2011). Using BioMark HD of Fluidigm, small difference in copy number could be identified which was approximately between six and seven copies of the specific gene (Jian et al., 2008). Wide-field fluorescence imaging by Digital PCR was used for analysis of single cells, organisms or molecules (Andrew et al., 2011).
Droplet digital PCR (ddPCR) ddPCR is used in phage-host interaction study (Tadmoret et al., 2011), fetal screening (Lo et al., 2007), biomarker analysis (Day et al., 2013), viral detection(Shen et al., 2011), prognostic monitoring (Belgrader et al., 2013), intracellular profiling (Sindelka et al., 2008), detection of GMO in food (Morisset et al., 2013), in high throughput sequencing (White et al., 2009), mitochondrial DNA alteration in Alzheimer disease (Podlesniy et al., 2013), and in mitochondrial DNA mutations (Taylor et al., 2014). QX100 Droplet Digital PCR system partitions the nucleic acid sample into approximately 20,000 droplets, with target and background DNA randomly distributed among them. After PCR amplification occurs, a reader determines which droplets contain a target (positive) and which do not (negative). ddPCR quantifies DNA copy number by partitioning PCR reactions into droplets (i.e. 20,000 separate reactions each containing a fluorescentlabeled probe) and then counting positive droplets, enabling enumeration of amplifiable molecules in the starting sample. The readout is the number of PCR plus and PCR minus droplets
Droplet digital PCR technology
DOI: 10.3109/19401736.2014.913168
standard curve and the concentration calculation is done based on Poisson algorithm which claims more accuracy and reproducibility (Lin, 2012).
Methodology Following standard protocol using TRIZOL reagent, 20 ng cDNA prepared. The ddPCR work flow starts by partitioning the TaqMan reaction mix containing cDNA into droplets in oil using droplet generator. PCR reaction mixtures of 20 mL volume comprising 1 ddPCR Master Mix (Bio-Rad-186-3010), relevant primer and probe mix (20) (900 nM each primer, 250 nM probe)
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from each reaction. The software calculates the concentration of target DNA as copies per microliter. The system uses microfluidic circuits and surfactant chemistries to divide a microliter mixture of sample and reagents into the monodisperse droplets, which support PCR amplification of single template molecules using homogeneous assay chemistries and workflows similar to those used for real-time PCR applications. An automated droplet flow cytometer reads each set of droplets after PCR at a rate of 32 wells per hour (Baker, 2012). Digital PCR has more applications in cancer research, mutation detection, prenatal diagnosis and copy number variation studies. The method uses no CT value, endogenous control or
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Figure 1. (A) Scatter plot showing positive and negative partions. (B) Histogram showing positive and negative partions. (C) Plot showing concentration (copies/microliter) based on poisson algorithm. (D) Plot showing events vs sample, demarking positive, negative and accepted droplets.
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P. Manoj
and DNA were prepared. Each 20 mL reaction was dispensed into a separate well of a disposable eight channel droplet generator cartridge (Bio-Rad DG8 catridges and gaskets, 186–3006). No Template Controls (NTC) containing 1 TE0.1 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) in place of DNA were also used in the reaction set up. 70 mL of droplet generation oil (186–3005) was then added into each of the corresponding oil wells before the chip was loaded into the droplet generator (QX100, Bio-Rad). The droplet generator applies a vacuum to each of the outlet wells to generate droplets in the eight channels simultaneously at a rate of 1000 droplets per channel per second until the complete 20 mL ddPCR mixture has been partitioned into the water-in-oil emulsion format. Each water-in-oil emulsion (40 mL) was transferred using multichannel pipette to separate wells in a 96well polypropylene plate (Eppendorf), sealed and amplified in a conventional thermal cycler (C1000 Touch Thermocycler-Bio rad). Two step thermo cycling protocol consisted of a 10 min activation period at 95 C followed by 40 cycles of 30 s at 94 C denaturation and 60 s at 60 C for combined annealing-extension at ramp rate set to 2.5 C/sec) ramp rate and a final 10 min inactivation step at 98 C. After thermal cycling, plates were heat sealed with aluminium sealer, then transferred to QX 100 droplet reader (Bio-Rad) containing droplet reader oil (186–3004), that employs an integrated auto sampler and fluidics to serially aspirate droplets from each well and stream them single-file, at a rate of 1500 droplets/second, past a two-color fluorescence detector sampled at a rate of 100 kHz on both FAM and VIC fluorescence channels. Discrimination between droplets that did not contain target (negatives) and those which did (positives) was achieved by applying a global fluorescence amplitude threshold in Quanta Soft (Bio-Rad), the software package provided with the ddPCR system for data acquisition and analysis. The droplet reader was calibrated once upon installation of the ddPCR system at the Institute RGCB, India. The simple calibration procedure for generation of an instrument-specific color compensation matrix was done and that was stored on the droplet reader and automatically applied to data to eliminate cross talk between FAM and VIC labeled probes. Quanta Soft uses a proprietary signal-processing algorithm to automatically perform droplet gating within each run. The threshold was set as the midpoint between the average fluorescence amplitude of positives and negative droplet clusters on each of the FAM and VIC channels.
Results and discussion ‘‘Quanta soft’’ application software (Bio-rad) was used for data analysis with default settings. A series of experiments were designed to detect dioeciousness of Piper longum plants for breeding purpose. cDNA was made from purified RNA of fresh leaf samples of male and female plants of piper, quantified, diluted, and used for ddPCR. Three replicates of each diluted DNA were prepared in eight-well plates using droplet generator cartridges. Calculation of copy number was based on Poisson distribution. The scatter diagram, and histogram, showed the positive and negative droplets of the target gene, from which copy number per microliter of sample was calculated by poisson equation. Events, number of positive droplets, negative droplets and accepted droplets were shown in graph (Figure 1). Multiplex reaction was also performed using FAM and VIC labeled probes for our control material with 2 target gene. Manual pippetting of template and primer/probes resulted in varying amplitude of positive droplets causing different signal to noise ratios and difficulty in setting a threshold for positive/negative droplets. NTC has shown low-level background indicating very less
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contamination during sample handling. Based on the number of positive partitions, the concentration was calculated using the formula: M ¼ ln [1[P/R]] copy number per droplet [9]. This calculation is based on the assumption that all the partitions are of equal volume. Poisson statistics are needed to account a positive PCR partition containing more than one molecule. The amount of template used for the experiment is an important factor in dPCR. Absorbance measurements of the DNA are taken at 260 nm. Concentration is calculated by dividing the copy number by the reaction volume, in which the size of the droplet is also to be considered for accuracy provided the droplets remain a uniform size even when the temperature fluctuates slightly. An accurate estimation of partition volume is important because different instruments may have different partition volumes. The sum of the partitions multiplied by partition volume will give the total volume of the reaction. DNA copy number concentration was around 1000 to 17,000. On binomial approximation 99.5% saturation of the droplets means 10 negative droplets in a ddPCR containing 20,000 droplets indicate between 10,200 and 110,000 copies of target DNA. Here the report is on a pilot study using plant specific primers against cDNA from different plants. Here we have used well identified male and female plant samples for the study and the experiment termed to be successful in identifying the sex of the plant samples, where FAM labelled primers shown amplification only from female plants and VIC labelled primers in male plants only. In this study only six primers were used and the experiment to be continued with more number of primers to generate sufficient data.
Conclusion Droplet Digital PCR has become an emerging tool for new applications and research. It is used in quantification of miRNAs, pathogens, in rare allele detection, and copy number variation detection. The accumulation of mt DNA mutations associated with aging, neuromuscular disorders and cancer are quantified through droplet digital PCR. The method overrides the variation of amplification, with high level of reliability and reproducibility. Since it is carried out in minute volumes and in single molecule concentrations, it has to be done with accuracy. It is an end point assay, and after PCR each partition will contain a negative or positive target of the nucleic acids which will be quantified by counting positive and negative reactions. As in qPCR inhibitors can also create false negatives by blocking the reaction to occur. Prior to the assay designing we should make sure that there is only one target. Multiple targets can be separated as different partitions in different wells. In case of rare allele detection, rates of false positives are very crucial. It may mask the number of true positives. Getting an estimate of the initial concentration of the template may help in making appropriate dilution of the sample. Droplet Digital PCR technology is highly reproducible with higher specificity sensitivity and accuracy, which can detect targets among fragments of DNA and has a unique ability to perform sensitive molecular measurements without the need of a standard curve. The conversion of real-time PCR analogue signal to binary output simplifies the measurement considerably. The major strength of ddPCR is the direct counting approach which provides precise, quantitative measurements without reference to a calibrator.
Declaration of interest The author declares no competing interests by conducting this study.
DOI: 10.3109/19401736.2014.913168
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