Method of isolating nucleic acids for long sequencing reads

Abstract

The invention is a novel method of isolating long nucleic acids from samples suitable for nucleic acid sequencing. The method is especially suitable for isolating low-concentration nucleic acids, e.g., viral nucleic acids, from clinical samples.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This patent application is a continuation of International Patent Application No. PCT / EP2017 / 069449 filed Aug. 1, 2017, which claims priority to and the benefit of U.S. Provisional Application No. 62 / 375,244, filed Aug. 15, 2016. Each of the above patent applications is incorporated herein by reference as if set forth in its entirety.FIELD OF THE INVENTION[0002]The invention relates to the field of nucleic acid isolation. More specifically, the invention relates to the field of isolating long nucleic acid molecules that can be used, for example, in sequencing applications.BACKGROUND OF THE INVENTION[0003]Existing methods of isolating nucleic acids result in breakage or chemical degradation of a portion of the nucleic acid resulting in recovery of short strands (<500 bp). The problem is especially severe for single-stranded nucleic acids, including RNA. At the same time, contemporary detection technologies, such as real-time quantitativ...

AI Technical Summary

Benefits of technology

The present patent describes novel methods for isolating nucleic acids from samples for long read sequencing purposes. The methods involve contacting the sample with a lysis buffer containing a reducing agent, detergent, carrier molecule, and protease, followed by contacting it with a nucleic acid-retaining separation matrix that binds the nucleic acid. The matrix is then washed and the nucleic acid eluted with a buffer. The eluate is then concentrated at least 2-fold. These methods can be used with a variety of samples, such as patient samples, and can also include steps to precipitate the sample and treat it with enzymes. The isolated nucleic acids can then be further used in downstream applications such as genomic sequencing.

Problems solved by technology

Existing methods of isolating nucleic acids result in breakage or chemical degradation of a portion of the nucleic acid resulting in recovery of short strands (<500 bp).

The problem is especially severe for single-stranded nucleic acids, including RNA.

Certain single-stranded nucleic acids, including viral RNA molecules are inherently more delicate and easily degraded as compared to DNA molecules during traditional nucleic acid extraction protocols.

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