Scaffolded Nucleic Acid Polymer Particles and Methods of Making and Using

Abstract

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

Description

BACKGROUNDIn order to generate sufficient signal for analysis, many applications in genomics and biomedical research require the conversion of nucleic acid molecules in a library into separate, or separable, libraries of amplicons of the molecules, e.g. Margulies et al, Nature 437: 376-380 (2005); Mitra et al, Nucleic Acids Research, 27: e34 (1999); Shendure et al, Science, 309: 1728-1732 (2005); Brenner et al, Proc. Natl. Acad. Sci., 97: 1665-1670 (2000); and the like. Several techniques have been used for making such conversions, including hybrid selection (e.g., Brenner et al, cited above); in-gel polymerase chain reaction (PCR) (e.g. Mitra et al, cited above); bridge amplification (e.g. Shapero et al, Genome Research, 11: 1926-1934 (2001)); and emulsion PCR (cmPCR) (e.g. Margulies et al, cited above). Most of these techniques employ particulate supports, such as beads, which spatially concentrate the amplicons for enhanced signal-to-noise ratios, as well as other benefits, such ...

AI Technical Summary

Benefits of technology

The present invention provides new methods and compositions for analyzing nucleic acids using porous microparticles with increased attachment capacity for polynucleotides. These particles have a larger surface area to volume ratio than spherical beads, which allows for more attachment of template molecules. The particles can have non-nucleoidie polymer networks attached to them, which further increases the number of attached polynucleotides. The particles can also have a smaller average nearest neighbor distance of the polynucleotides, which improves the accuracy of analysis. The invention also provides amplicon libraries with clonal populations of polynucleotides attached to non-nucleosidie polymer networks. The libraries have a low coefficient of variation and can be enriched to improve accuracy. The invention also includes methods of making monodisperse populations of gel particles and using them to make amplicon libraries without an emulsion reaction.

Problems solved by technology

In some cases, amplicons are either in a planar format (e.g. Mitra et al, cited above; Adessi et al, Nucleic Acids Research, 28: e87 (2000)), which limits ease of manipulation and / or reagent access, or the amplicons are on bead surfaces, which lack sufficient fragment density or concentration for adequate signal-to-noise ratios.

Such emulsion reactions are labor intensive and require a high degree of expertise, which significantly increases costs.

However, such particulates are limited in that they are typically produced with widely varying size distributions, particularly at lower size ranges, e.g. less than about 30 μm, which makes them unsuitable for many exacting analytical applications, such as large scale DNA sequencing.

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