Methods of nucleic acid sample preparation

Inventors

Stahl, Joshua • Myers, Jason • Culver, Brady • Kudlow, Brian

Assignees

Labcorp Holdings Inc

Abstract

Aspects of the technology disclosed herein relate to methods of 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 methods of preparing nucleic acids for analysis using a capture moiety modified nucleotide associated with an adapter-based ligation workflow. A double-stranded nucleic acid comprising a target nucleotide sequence receives one or more nucleotides at a 3′ end, where at least one nucleotide is a capture moiety modified nucleotide, and an adapter nucleic acid is ligated to at least one strand to produce a ligation product comprising the capture moiety modified nucleotides.

The prepared ligation product is captured by contacting it with a binding partner of the capture moiety modified nucleotide, thereby providing an immobilized ligation product in some embodiments. In certain embodiments, the ligation product is separated from the binding partner and amplified to provide amplicons, including amplification by a polymerase chain reaction using a first target-specific primer and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.

The method extends to preparing nucleic acids from a plurality of double-stranded nucleic acids comprising different target nucleotide sequences. The approach adds capture moiety modified nucleotides to 3′ ends of each double-stranded nucleic acid, ligates an adapter to generate a plurality of ligation products, captures the plurality of ligation products using the binding partner, and amplifies the plurality of ligation products to provide a plurality of amplicons.

Claims Coverage

The consolidated content identifies four independent claims. Across these claims, the inventive features center on 3′-end addition of capture moiety modified nucleotides, adapter ligation to form capture-modified ligation products, capture using a binding partner, and, in some claims, separation and amplification to provide amplicons, including parallel processing of multiple different target nucleotide sequences.

Across the independent claims, the coverage focuses on preparing nucleic acids for analysis by adding capture moiety modified nucleotides to 3′ ends of double-stranded nucleic acids, ligating adapters to create capture moiety-containing ligation products, capturing those products using a binding partner of the capture moiety, and, in certain claims, separating and amplifying to provide amplicons or directly amplifying after capture for multiple target nucleotide sequences.

Stated Advantages

Documented Applications