Methods of preparing nucleic acids for sequencing

Inventors

Iafrate, Anthony John • Le, Long Phi • Zheng, Zongli • Myers, Jason • Stahl, Joshua

Assignees

General Hospital Corp • Labcorp Holdings Inc

Abstract

Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

Core Innovation

The invention relates to a method of determining the nucleotide sequence contiguous to a known target nucleotide sequence. It uses a target nucleic acid molecule comprising the known target nucleotide sequence and an initial target-specific primer that specifically anneals to the known target nucleotide sequence. The method includes template-dependent extension primed by the hybridized initial target-specific primer to produce an extension product that uses the target nucleic acid molecule as a template.

The extension product is contacted with a plurality of tailed random primers under hybridization conditions, and a second template-dependent extension reaction is performed. The second extension is primed by a hybridized tailed random primer and uses the extension product of the first extension reaction as a template. Each tailed random primer comprises a common sequence and a 3'-end comprising about 3 to about 15 random nucleotides, enabling sequencing of nucleotides contiguous to the known target sequence.

To enable sequencing-compatible amplification and readout of the contiguous nucleotides, a portion of the extension product from the second extension is amplified using a first tail primer and a first target-specific primer. The first target-specific primer comprises a 3'-portion that specifically anneals to a portion of the known target nucleotide sequence and is nested with respect to the initial target-specific primer. The first tail primer comprises a nucleic acid sequence identical to a portion of the common sequence, and the amplification product is sequenced using a first or second sequencing primer.

Claims Coverage

The independent claim is clm-00001. It includes six inventive features: initial target-specific extension, extension with a plurality of tailed random primers, the tailed random primer structure, nested target-specific amplification, tail-primer amplification, and sequencing of the amplification product.

The coverage centers on contiguous-sequence determination from a known target using initial target-specific extension, extension primed by a plurality of tailed random primers with a common sequence and a 3'-random end, amplification with a tail primer identical to the common sequence portion and a nested target-specific primer, and sequencing of the amplification product. Dependent claims further narrow the tailed random primers with barcode/index sequences, specify a nesting distance constraint, and restrict tailed random primers to naturally occurring nucleotides.

Stated Advantages

Documented Applications