Detecting low levels of specific nucleic acid target

Transcription

1 Application Note Detection of Rare Nucleic Acid Targets using the Constellation Digital PCR (dpcr) System By Douglas Roberts, Formulatrix Detecting low levels of specific nucleic acid target sequences is of crucial importance to a variety of applications. For example, accurate and reliable measurement of molecules making up a small fraction of the total population is vital when judging the efficacy of eradication treatment 1 by identifying pathogen reservoirs in host tissue. Similarly, detecting viral or bacterial load to measure host response is helpful when predicting the future course of a virus- or bacteria-borne disease 2. The same nucleic acid identification is necessary for detecting early events of tumor progression 3 ; during prenatal screening that uses free fetal DNA in maternal background 4 ; detecting the presence of GMO material in food products 5 ; and for the quantitation of rare transcribed RNA species 6. Digital PCR is a highly accurate technique for the absolute quantitation of nucleic acid targets. It can also be used to detect the presence of rare target molecules. This application note describes the use of the Formulatrix Constellation Digital PCR system to detect and quantitate the presence of a synthetic target spiked into background DNA at various levels. The tests revealed accurate detection a single copy of target DNA in 17 µl of tested sample. Furthermore, rare target was detected in 75 ng/µl of background DNA, making the Constellation a valuable research tool for any of the above applications. touchscreen interface make the entire process seamless - from priming through sealing, imaging, and data analysis. METHODS AND MATERIALS Integrated DNA Technologies synthetic target from IDT containing target amplicons for both mouse hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyltransferase (HPRT). HMBS (HEX) and HPRT (FAM) PrimeTime qpcr assays were then synthesized by Integrated DNA Technologies at the default primer/probe ratio of 2:1, and re-suspended according to the manufacturer's instructions to generate a 2x stock. Assays were added to PCR reactions at a final concentration of 1x (unless otherwise indicated). All experiments used TaqMan Fast Advanced Master Mix (Life Technologies, Cat. No ) according to manufacturer's instructions. Salmon sperm genomic DNA (Invitrogen Cat. No ) was added to the ABOUT THE FORMULATRIX CONSTELLATION The Constellation is an intuitive, user-friendly, low-cost, high-throughput dpcr device. Based on a proprietary microtiter plate that conforms to SBS standards, the Constellation enables sample loading with conventional manual or robotic pipetting tools. The initial version of this plate contains 96 individual sample wells, each of which is processed by the Constellation through priming and sealing steps into 496 separate partitions. After thermal cycling on conventional equipment, the plates are read by the Constellation using up to four separate optical channels. Simply, clean software and a Formulatrix Constellation digital PCR system 1 Copyright 214

2 reactions at the indicated final concentrations where applicable. Template Dilution Expected copies (all wells) FAM total copies VIC total copies Samples were run in replicate wells on a single Constellation plate. Partitions were primed using the Constellation instrument and the entire plate was thermal cycled either with a 3 second hot start at 95 C, followed by 4 cycles of a 5 second, 95 C denaturation step, paired with a 3 second, 6 C annealing step, or altered cycling conditions depending on the specific experiment. Plates were then imaged by the Constellation for ROX, FAM and HEX using standard imaging protocols. The Constellation software determined copy number quantities for each sample for individual wells and reported the average and standard deviation of technical replicates. RESULTS Test 1: The Constellation Quantifies 1-2 Targets per 17.5 ul Sample The Constellation digital PCR system tested five samples of known levels of spiked-in synthetic target in a constant background of 1 ng/µl salmon sperm DNA (see Table 1). Eight-well replicate reactions were cycled with a 3 second hot start at 95 C, followed by 4 cycles of a 5 second, 95 C denaturation step paired with a 3 second annealing step at 6 C. 1: : :1, :4, NTC 1 Table 1. Experimental replicate information, expected numbers of target copies, and actual copies observed. For the most concentrated samples, expected totals were either 211 or 21.1 copies. For the 1:2 sample, about 26 copies of HPRT or 24 copies HMBS were observed. For the 1:2 sample, 23 and 25 copies were observed. This is represented graphically in Fig. 1a. For the rarer target samples, the 1:1, dilution was expected to yield 4.22 targets, and was observed to have seven HPRT or six HMBS, respectively (see Table 1 and Fig. 1b). For the 1:4, dilution, the number of expected copies for the 4, partitions tested was approximately one. Three HPRT and two HMBS copies were observed. As no rare target detection experiment can be interpreted without knowledge of false positive frequency, eight replicates of the HPRT/HMBS assay were tested as No Template Control (NTC) samples. A single positive was 3 Rare Target Detection 2 Rare Target Detection Template Count 2 1 Template Count :2 1:2 1:1, 1:4, NTC Template Dilution 1:2 1:2 1:1, 1:4, NTC Template Dilution FAM total copies VIC total copies FAM total copies VIC total copies Figure 1a. Constellation dpcr quantification of spiked-in synthetic target using IDT 5 nuclease assays designed against HPRT (FAM) or HMBS (HEX). Figure 1b. Y-axis zoom of Figure 1a. Low level target detection of spikedin synthetic target using the Constellation dpcr system. 2 Copyright 214

3 FAM 4 cycles FAM 6 cycles 3, ng/µl 1,5 ng/µl 3 ng/µl 15 ng/µl 3 ng/µl 15 ng/µl 7.5 ng/µl Figure 2a. 4-cycle and 6-cycle endpoint images of Constellation wells containing a constant amount of target in varying amounts of background DNA. observed in the HPRT partitions, and zero positives were observed in the HMBS partitions. Since the 95% confidence intervals for the 1:4, dilutions are non-overlapping with those of the NTC samples, the detection of the targets in the 1:4 sample is statistically significant. This finding represents the accurate quantitation of 1-2 target molecules per 17.6 µl of sample tested, or 1.76 µg of background DNA, or about 5x15 genomes. The level of rare target detection by the Constellation system can be extended by running more replicate wells, or by using more concentrated template samples. However, when running more replicates, the question arises as to how to avoid the false positives lower limit. Likewise, the amount of background DNA present can compromise the limit of rare target detection. The Effect of Background DNA on Target Detection To determine the effect of background DNA on target detection in the Constellation system, a sample containing 35 copies/µl synthetic target was assayed for the presence of HPRT or HMBS target in a background of salmon sperm DNA of varying concentrations. Duplicate reaction wells containing 7.5 ng/µl, 15 ng/µl, 3 ng/µl, 75 ng/µl, 15 ng/µl, 3 ng/µl,, 15 ng/µl or 3 ng/µl were cycled with a 3 second hot start at 95 C, followed by 4 cycles of a 5 second, 95 C denaturation step paired with a 3 second, 6 C annealing step. Following an initial imaging session using the standard Constellation protocol, an additional 2 cycles were conducted using a 15 second, 95 C denaturation step, paired with a 6 second, 6 C annealing step, to enable any further amplification reactions to run to completion. FAM images for both 4- and 6-cycle reactions for each duplicate pair of background salmon DNA concentration are shown in Figure 2a. Clear, uninhibited reactions are observed at 4 cycles for all reactions containing background DNA levels of 15 ng/µl or less. In contrast, no amplification is observed at 4 cycles with samples containing greater than 3 ng/µl of background DNA. Such a sharp transition between accurate detection and no amplification has been observed previously when assessing the effects of background DNA on rare target detection 7, though in the referenced study, inhibition was reported with background DNA concentrations as low as. The 4 cycle FAM image for the background level at 3 ng/µl displays a recognizable pattern of amplification, but one that is clearly much less efficient than reactions containing less background DNA. Reaction observation following the 6 cycles was indicative that the 3 ng/µl reactions had achieved completion, as the template quantity obtained matched that of reactions with lower background DNA; the HMBS (HEX) assay images depicted a similar amplification pattern (data not shown). 3 Copyright 214

4 Template Copies Effect of Salmon Sperm DNA on dpcr Assay Quantitation Salmon sperm DNA in Reaction, ng/µl FAM average VIC average Figure 2b. 4-cycle endpoint analysis of the effect of background DNA on target quantitation by digital PCR using the Constellation. As expected, for each sample with complete amplification at 4 cycles, the template quantities determined for the HPRT FAM assay were consistent and independent of the background concentration level (see Figure 2b). Further, additional sets of 2 cycles of conditions identical to those used for cycles 41-6 failed to demonstrate any amplification in background DNA concentrations above 3 ng/µl (data not shown). Since the prior experiment demonstrated that the addition of background DNA significantly compromised assay performance, but could be partially restored by cycling conditions, it implies that the PCR process was being affected by one or more factors. The most likely of these factors involves the assembly efficiency of either primers or polymerase into replication-competent assemblies, or the sequestration of primers and/or probe in non-productive replication complexes. Overcoming the Inhibitory Effect of Background DNA These factors, coupled with the observation that there was a distinct, abrupt transition in assay performance between 3 ng/µl and suggests a need for higher resolution examination within this range of background DNA concentrations. Additional experiments were thus conducted to more precisely locate and outline FAM HEX 625 ng/µl 45 ng/µl 3 ng/µl 15 ng/µl 2x assay 5x assay 2x Pol 4x Pol Figure 3a. 4-cycle endpoint image of a high-resolution examination of the extent of PCR inhibition by background DNA and examination of additional reaction conditions. 4 Copyright 214 COA-2R114

5 this transition region and to explore reaction conditions that might ameliorate the previous-inhibitory levels of background DNA. To further define the limit of background inhibition on digital PCR in the Constellation system, a sample containing 35 copies/µl synthetic target was assayed for the presence of HPRT or HMBS target in a background of salmon sperm DNA of finely varying concentration. Duplicate reaction wells containing, 15 ng/µl, 3 ng/µl, 45 ng/µl, 625 ng/µl, and of background DNA, or combinations of background DNA, and either 2x or 5x assay or 2x or 4x DNA polymerase, were cycled with a 6 second hot start at 95 C, followed by 4 cycles of a 15 second, 95 C denaturation step, paired with a 6 second, 6 C annealing step. Following an initial imaging session using the standard Constellation protocol, an additional 2 cycles for a total of 6 cycles were conducted using a 2 second, 95 C denaturation step, paired with a 9 second, 6 C annealing step, to allow any further amplification reactions to run to completion. FAM and HEX images for 4-cycle reactions for each duplicate pair of reaction conditions are shown in Figure 3a. Immediately evident from the examination of the 4- cycle images for both the HPRT (FAM) and HMBS (HEX) assays is that both the upper limit of obtaining detectable reaction products has been extended above the previously-observed background limit of 3 ng/µl, and that a background concentration of genomic DNA previously observed to be completely inhibitory to PCR can be finessed to support productive amplification. Both the 45 ng/µl and 625 ng/µl samples show detectable signal and distinct negative partitions. However, both reaction conditions, as well as those of the 3 ng/µl reactions, yield insufficient positive signal for reliable quantitation (see Fig. 3b). Similarly, the addition of 5x assay or 2x or 4x polymerase is sufficient to overcome the inhibitory effects of background DNA, but not such that accurate template quantitation can be achieved. 1 Effect of Background DNA 8 Template Count ng/µl 3 ng/µl 45 ng/µl 625 ng/µl 2x assay 5x assay 2x Pol 4x Pol Reaction Condition FAM average VIC average Figure 3b. 4-cycle endpoint results examining the extent of PCR inhibition by background DNA and examination of additional reaction conditions. 5 Copyright 214

6 FAM HEX 625 ng/µl 45 ng/µl 3 ng/µl 15 ng/µl 2x assay 5x assay 2x Pol 4x Pol Figure 3c. 6-cycle endpoint image examining the extent of PCR inhibition by background DNA and examination of additional reaction conditions. The observation that cycling parameters and increased assay and/or polymerase can rescue reactions inhibited by increased levels of background DNA implies an inhibitory mechanism whereby active replication complexes fail to assemble in a timely and productive manner. Additional Cycling Provides Additional Complete Reactions Despite Background DNA To obtain accurate quantitation of samples displaying incomplete reactions after 4 cycles, an additional 2 cycles were conducted using the same denaturation and annealing conditions as the first 4 cycles. Endpoint images are shown in Figure 3c. Reaction conditions that were incomplete after 4 cycles in Figure 3a are clearly closer to completion in Figure 3c, although not all achieve identical levels of endpoint fluorescence when compared to reactions containing lower levels of background DNA. Whether this is a reflection of these PCR reactions not achieving equivalent endpoint levels or a more generalized non-specific effect on reporter fluorescence was not determined. Similarly, the reactions containing background DNA and additional reaction components show additional progression to completion. However, in each case where additional reaction components were observed to allow for productive amplification in a level of background DNA previously observed to be completely inhibitory, none of the conditions tested contained positive partitions at a level equivalent to less inhibited samples (see Fig. 3d). These results depict a clear indicator that while some level of inhibition can be alleviated by altered reaction components utilizing the cycling conditions presented, the level of background DNA is still inhibitory to an extent that affects accurate quantitation. Due to the nature of PCR, assay efficiency -- and hence the levels of background DNA which might be tolerated by a specific assay -- are likely to be specific to each individual assay. Thus, under the cycling conditions and assay tested, the maximum level of background reliably tolerated is 45 ng/µl, and it is probable that if an extension of the tolerable range is required for an application, reaction conditions may be optimized. The Effect of Background DNA on Rare Target Detection Because all previous background tolerance experiments were performed using high levels of template, it was necessary to ensure that background DNA levels demonstrated to provide accurate quantitation of moderately high levels of target would also give accurate results for rare targets. To assess the effect of background DNA on rare target detection, eight replicates of a seven copy/µl and a No Template Control (NTC) were tested in the presence of either 15 ng/µl, 45 ng/µl or salmon sperm DNA. Reactions were cycled with a 6 second hot start at 95 C followed by 6 cycles of a 15 second, 95 C denaturation step, paired with a 6 second, 6 C annealing step. 6 Copyright 214

7 1 Effect of Background DNA 8 Template Count ng/µl 3 ng/µl 45 ng/µl 625 ng/µl 2x assay 5x assay 2x Pol 4x Pol Reaction Condition FAM average VIC average Figure 3d. 6-cycle endpoint analysis of the effect of DNA background on target quantitation by digital PCR in the Constellation system. The results of the rare target detection are shown in figure 4a. The low levels of target were detected accurately, precisely and reproducibly in the salmon DNA background samples of 15 ng/µl and 45 ng/µl. As in aforementioned experiments, under standard cycling and other reaction conditions, salmon sperm background DNA inhibited detection of the target. Since the level of false positives can affect the utility of digital PCR as a tool for detecting low levels of target 1, the results of the NTC reactions are shown in figure 4b. For all levels of background DNA examined, the HPRT (FAM) assay had at least one positive partition per 3,986 partitions, while the HMBS (HEX) assay yielded only positive NTC partitions in the 45 ng/µl sample. In every case, the level of false positives was statistically distinguishable from the samples containing low levels of target. Low Copy Target NTC Template Quantity HPRT (FAM) HMBS (VIC) Template Quantity HPRT (FAM) HMBS (VIC) 15 ng/µl 45 ng/µl Background DNA Concentration Figure 4a. 6-cycle endpoint analysis of the effect of DNA background on low-level target quantitation by digital PCR in the Constellation system were cycled with a 6 second hot start at 95 C followed by 4 cycles of a 15 second 95 C denaturation step, paired with a 6 second, 6 C annealing step. 15 ng/µl 45 ng/µl Background DNA Concentration Figure 4a. 6-cycle endpoint analysis of the effect of DNA background on NTC quantitation by digital PCR in the Constellation system. 7 Copyright 214

8 CONCLUSIONS The Constellation digital PCR system detects target nucleic acid in samples that contain as few as 1-2 copies of target nucleic acid per 17.6 µl in a background of approximately 1.76 µg cellular DNA equivalents. Further, accurate quantitation of both 35 copies/µl samples and 7.5 copies/µl samples of synthetic template using standard cycling and reaction conditions is possible at levels of 3 ng/µl, extendable to 625- via altered cycling and/or reaction conditions, making it the most sensitive dpcr system available. A study released about a comparable digital PCR system shows that the Constellation resolves target nucleic acid in the presence of background DNA with at least 1 times the sensitivity of other systems 7. Strain et al. reported that the effect of background DNA under the testing conditions used in their experiments had a sharp transition from accurate amplification to complete reaction failure between µg of background DNA. Since 2 µl reaction volume was used in the experiment, levels of background DNA were equivalent to an inhibitory level of between 5 ng/µl and, which is 1/1th that attainable with the Constellation. REFERENCES 1. Kiselinova M et al., Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA. 214, PLoS ONE 9(1): e Sedlak RH, et al., Clinical utility of droplet digital PCR for human cytomegalovirus. 214, J Clin Microbiol. Aug;52(8): Cochran RL, et al. (214), Analysis of BRCA2 loss of heterozygosity in tumor tissue using droplet digital polymerase chain reaction. 214, Hum Pathol. Jul;45(7): Barrett AN and LS Chitty. Developing noninvasive diagnosis for single-gene disorders: the role of digital PCR. 214, Methods Mol. Biol. 116: Morisset D et al., Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR. 213, PLoS ONE 8(5): e Lacobucci, L et al. (214), Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positive acute lymphoblastic leukemia in complete response. Leuk Res. 38(5): Strain MC et al. (213), Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One. 8(4):e Formulatrix 1 DeAngelo Drive Bedford, MA 173 USA Phone: Copyright 214