The foregoing application, and all documents cited therein or during their prosecution (“appln cited documents”) and documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods for quantitating nucleic acids, advantageously double stranded nucleic acids, such as double stranded DNA.

BACKGROUND OF THE INVENTION

Single-gene studies, metagenomic assemblies, and the genome sequences of a limited number of cultured isolates are not a sufficient basis on which to accurately model the responses of natural microbial networks or engineer the function of artificial communities. For example, gene catalogs and composite genomes assembled from metagenomic data do not presently distinguish between genes that are tightly coupled within the context of the same organism and genes that are coupled across different organisms. This is a critical limitation, because only gene products encoded by the same organism can freely come into contact with one another to form complexes, drive signaling pathways, or carry out multi-step enzymatic transformations of diffusible substrates at maximum efficiency. A systems-level predictive understanding of microbial physiology absolutely demands the interpretation of genes and pathways in a full genomic context. Furthermore, individual organisms (indeed single cells) encoding full genomes are the basic replicating unit of biology and an important unit of evolutionary selection, factors that cannot be ignored in understanding the development of microbial networks in larger populations as a function of time.

In practical terms for single microbial cell sequencing, this means a million-fold amplification of the DNA present at the time of cell selection is required. Such high fold-amplification from sub-nanogram samples (Dean, et al., 2002, Proceedings of the National Academy of Sciences 99: 5261) and individual bacteria (Raghunathan, et al., 2005, Applied and Environmental Microbiology 71: 3342-3347) with good representation of the genome were first achieved by the multiple displacement amplification (MDA) whole-genome amplification (WGA) chemistry, but produced material with undesirable characteristics such as uneven representation and dislocated sequences. Nonetheless, investigators shortly succeeded in assembling shotgun sequence reads from single WGA-amplifiedE. coliand Prochlorococcus (Zhang, et al., 2006, Nat Biotechnol 24: 680-686), TM7 (Marcy, et al., 2007, Proc Natl Acad Sci USA 104: 11889-11894), and sequencing multiple genes fromE. coli(Marcy, et al., 2007, PLoS Genet 3: 1702-1708), single marine bacteria (Stepanauskas & Sieracki, 2007, Proc Natl Acad Sci USA 104: 9052-9057), and soil and cultivated archaea (Kvist, et al., 2007, Applied microbiology and biotechnology 74: 926-935). Since the development of MDA, many other WGA methods have been developed and successfully applied to single cells (Blainey 2013, Cai & Walsh et al, 2012).

MDA is the WGA method that has been most commonly applied in single-cell sequencing of microbes. MDA works by the extension of 6-mer 3′-protected random primers on the DNA template (Dean, et al., 2001, Genome Res 11: 1095-1099). In MDA, a polymerase with strong strand displacement activity such as phi29 DNA polymerase or Bst DNA polymerase creates and displaces overlapping synthesis products from the template as single-stranded DNA under isothermal conditions (Dean, et al., 2001, Genome Res 11: 1095-1099; Zhang, et al., 2001, Mol Diagn 6: 141-150; Aviel-Ronen, et al., 2006, BMC Genomics 7). The displaced single-stranded DNA is a substrate for further priming and synthesis (Dean, et al., 2001, Genome Res 11: 1095-1099; Zhang, et al., 2001, Mol Diagn 6: 141-150). Phi29 DNA polymerase is typically specified for MDA due to its high accuracy owing to 3′-5′ exonuclease-mediated proofreading and exceptionally strong processivity in strand displacement synthesis, which can exceed 10,000 nt (Mellado, et al., 1980, Virology 104: 84-96; Blanco & Salas, 1984, Proceedings of the National Academy of Sciences 81: 5325; Blanco, et al., 1989, Journal of Biological Chemistry 264: 8935-8940; Morin, et al., 2012, Proceedings of the National Academy of Sciences 109: 8115-8120). This property of the polymerase evens out amplification on shorter genomic distances to produce high molecular weight products with more uniform amplification across the template than purely PCR-based methods, which typically produce products shorter than 1000 nt and exhibit greater amplification bias (Dean, et al., 2002, Proceedings of the National Academy of Sciences 99: 5261). In late 2012, one vendor started marketing a MDA kit that is decontaminated with ultraviolet light treatment and includes a mutant enzyme claimed to improve amplification uniformity and chimera performance (Qiagen REPLI-g Single Cell).

Given the concentration of contaminating fragments present in commercial WGA reagents (varying from 5 to 50 fragments per reaction microliter in the enzyme alone), volume reduction by itself does not necessarily eliminate reagent contamination (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). For example, even driving reaction volumes down to the low nanoliter range, a significant fraction of reactions are still expected to carry contaminates from the reagents. Thus it is necessary to either inactivate contaminates in the reagents or produce reagent sets that are free of contamination. Contaminates in commercial WGA kits have been successfully suppressed by UV exposure with acceptable post-treatment amplification performance (Zhang, et al., 2006, Nat Biotechnol 24: 680-686; Woyke, et al., 2011, Plos One 6: e26161). Alternatively, reagents for background-free WGA can be produced in batch processes utilizing disposable plasticware produced from virgin materials (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). Irrespective of the clean-up approach taken, a key capability for validating reagent lots and clean-up procedures is a rapid assay for WGA activity and contamination. To be useful, the contamination assay must be both quantitative and extremely sensitive, such that different lots or treatments can be evaluated comparatively. QPCR is insufficiently sensitive since only contaminant molecules with an intact sequence locus matching specific PCR primers can be detected. For example, a PCR assay for the small subunit ribosomal RNA gene misses thousands of contaminant fragments arising from bacterial genomic DNA for every fragment molecule detected. Alternatively, the digital WGA (dWGA, eg digital MDA or dMDA) assay can be used for quantitation down to a few attograms of degraded genomic DNA per microliter (Blainey & Quake, 2011, Nucleic Acids Res 39: e19). dWGA is compatible with a variety of off-the-shelf platforms engineered for digital PCR (Baker, 2012, Nature methods 9: 541-544).

There is a need for improved methods for detecting contamination in reagents as well as improved methods for quantitating DNA.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

This invention fulfills a need for improved methods for detecting contamination in WGA reagents as well as improved methods for quantitating DNA.

Technological advances improve the reliability and throughput of single-cell WGA and sequencing. The contamination of reagents with bacterial DNA that plagued early efforts in single-cell microbial genomics is now well-understood, and effective measures to quantify, control, and eliminate such contamination are disclosed herein. The elimination of background amplification not only yields clean datasets, but also allows the facile application of screens for successful WGA reactions. The move to lower-volume WGA reaction platforms further reduces the impact of contaminates and WGA reagent costs, opening the way to massive increases in reaction throughput.

The key drivers of new technology for single-cell genomics are advances in throughput, integration of isolation and WGA with selection and sequencing procedures, improvements in more uniform representation of template sequences in the WGA product, fewer artifact sequences such as chimeras, expanded sample/cell type compatibility, and contextualization of single-cell genome data by in-line collection of other information about the processed cells (eg phenotyping, imaging, RNA, protein, metabolite analyses). The ability to obtain sequence data from individual cells from a known biological setting or with a known history (e.g. interaction with other cells prior to the analysis) is especially informative. Because sample preparation costs already dominate single-cell microbial genomics workflows today, the most impactful advances will scale to large numbers of cells at reasonable cost.

The present invention relates to methods of quantifying nucleic acids which may comprise (a) contacting a sample to be tested with whole-genome amplification (WGA) reaction components and a dye molecule to form a reaction sample, wherein the dye molecule detects the nucleic acid; (b) partitioning the reaction sample wherein each partitioned reaction sample corresponds to a single reaction; (c) allowing the reaction to occur in the partitioned reaction sample and (d) determining the number of partitioned reaction samples having the dye molecule, wherein the dye molecule indicates the presence of the nucleic acid; thereby quantifying the nucleic acid is quantitated in the sample to be tested.

Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

