Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.
Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.
Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of ∼90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.
Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.
SummaryBlue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the ®rst 30 min of irradiation by a mechanism that depends on the phototropin 1 (phot1) photoreceptor. The cryptochrome 1 (cry1) photoreceptor begins to exert control after 30 min. To identify genes responsible for the cry1 phase of growth inhibition, mRNA expression pro®les of cry1 and wild-type seedlings were compared using DNA microarrays. Of the roughly 420 genes found to be differentially expressed at the point of cry1 response incipience, approximately half were expressed higher and half lower in cry1 relative to the wild type. Many of the cry1-dependent genes encoded kinases, transcription factors, cell cycle regulators, cell wall metabolism enzymes, gibberellic acid (GA) biosynthesis enzymes, and auxin response factors. High-resolution growth studies supported the hypothesis that genes in the last two categories were indeed relevant to cry1-mediated growth control. Inhibiting GA 4 biosynthesis with a 3b-hydroxylase inhibitor (Ca-prohexadione) restored wild-type response kinetics in cry1 and completely suppressed its long-hypocotyl phenotype in blue light. Co-treatment of cry1 seedlings with Ca-prohexadione plus GA 4 completely reversed the effects of the inhibitor, restoring the long-hypocotyl phenotype typical of the mutant. Treatment of wild-type seedlings with GA 4 was not suf®cient to phenocopy cry1 seedlings, but cotreatment with IAA plus GA 4 produced cry1-like growth kinetics for a period of approximately 5 h. The genomic and physiological data together indicate that blue light acting through cry1 quickly affects the expression of many genes, a subset of which suppresses stem growth by repressing GA and auxin levels and/or sensitivity.
We have identified three major blocks of amino acid homology shared by the transit peptides of two nuclear‐encoded chloroplast proteins, the light‐harvesting chlorophyll a/b‐protein (LHCP) II of the thylakoid membrane and the small subunit (SSU) of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) of the stroma. These previously unrecognized homology blocks lie at the beginning, middle and end of both transit sequences, and are separated by differing lengths of unshared (interblock) sequence in the two proteins. These interblocks may be dispensible or they might confer a specific property on the individual proteins, such as facilitating proper compartmentalization within the chloroplast. We propose that these three shared sequence elements form a common framework in transit‐bearing chloroplast precursors which mediates the common functions performed by each transit peptide. Ferredoxin, the only other such nuclear‐encoded protein for which a published transit sequence exists, conforms to the predictions of this hypothesis. These findings stand in contrast to mitochondrial leader sequences and the well‐studied signal peptides of secretory and certain integral membrane proteins in which no such framework has been observed.
Background: A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.
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