Methods of nucleic acid sample preparation for analysis of cell-free DNA

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, e.g, cfDNA. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

Core Innovation

The invention provides a method of preparing nucleic acids for analysis by adding one or more nucleotides to a 3′ end of a double-stranded nucleic acid comprising a target nucleotide sequence. The double-stranded nucleic acid is obtained from cell-free DNA, and at least one of the one or more nucleotides is a capture moiety modified nucleotide. The capture moiety modified nucleotide enables downstream capture of a ligation product using a binding partner of the capture moiety.

An adapter nucleic acid is ligated to the double-stranded nucleic acid to which the capture moiety modified nucleotide has been added to produce a ligation product. A sequence of one or more nucleotides at a 3′ end of the adapter nucleic acid is complementary with the one or more nucleotides added to the 3′ end of the double-stranded nucleic acid. The ligation product is captured by contacting the ligation product with a binding partner of the capture moiety of the capture moiety modified nucleotide.

For cfRNA, the method includes preparing a cDNA by randomly-primed first strand synthesis using cell-free RNA as template and second strand synthesis using the product of the randomly-primed first strand synthesis. The cDNA is end repaired to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence, followed by washing. One or more nucleotides are added to the 3′ end as capture moiety modified nucleotides, then an adapter is ligated using a ligatable duplex portion and an overhang sequence complementary to the one or more nucleotides added to the 3′ end.

After adapter ligation, nucleic acids are amplified by polymerase chain reaction using a first target-specific primer and a first adapter primer, and then amplified again using a second adapter primer and a second target-specific primer that is nested relative to the first target-specific primer. The workflow is described for sequencing upstream/downstream of known targets with high specificity/sensitivity.

Claims Coverage

Two independent claims are present. They center on adding a capture moiety modified nucleotide at a 3′ end, ligating an adapter nucleic acid through complementary 3′ or overhang sequences, capturing the ligation product via a binding partner of the capture moiety, and for cfRNA, generating blunt-ended double-stranded cDNA and using nested PCR with target-specific and adapter primers.

The inventive scope is anchored in adding a capture moiety modified nucleotide at a 3′ end of a target-containing cell-free derived double-stranded nucleic acid, ligating an adapter nucleic acid using complementary 3′ or overhang sequences to form a ligation product, capturing the ligation product via a binding partner of the capture moiety, and for cfRNA, generating blunt-ended double-stranded cDNA and using nested PCR with target-specific and adapter primers.

Stated Advantages

Documented Applications