Methods of Making Libraries of Nucleic Acids Using Porous Particles

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

[0001]This is a continuation-in-part of U.S. patent application Ser. Nos. 12 / 474,897 and 12 / 475,311 both filed 29 May 2009, and claims priority under U.S. provisional applications Ser. No. 61 / 263,734 filed 23 Nov. 2009; Ser. No. 61 / 291,788 filed 31 Dec. 2009; and Ser. No. 61 / 297,203 filed 21 Jan. 2010. All of the foregoing applications are incorporated by reference in their entireties.BACKGROUND[0002]In 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: c34 (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...

AI Technical Summary

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: c87 (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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