Patent ID: 11952613
Assignee: PHILLIP N. GRAY
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 2:
3. A method that attaches unique molecular identifiers (UMIs) and adapter sequences, and selectively captures non-denatured double stranded nucleic acid sequences simultaneously using the library of Dual Adapter molecules species of claim 2, the method comprising:
a. providing a reaction mixture comprising a nuclease activity and the library of dual adapter molecules of claim 2, and generating single stranded regions at the ends of the dual adapter molecules;
b. optionally providing a reaction mixture comprising a nuclease activity and population of target nucleic acids (optionally DNA, cDNA and/or RNA molecules), and generating single stranded regions at the ends of the target nucleic acid molecules;
c. providing a reaction mixture comprising a library of Dual Adapter molecules of claim 2 that comprise single-stranded HR1 and HR2 domains, a nucleic acid sample that comprises a population of target nucleic acids, optionally DNA, cDNA and/or RNA molecules, that optionally comprise single-stranded ends, a DNA polymerase activity, a nuclease activity and a DNA ligase activity, wherein the reaction mixture is incubated under conditions that facilitate hybridization between single-stranded complementary regions of the Dual Adapter molecules-HR1:HR2 sequences and target nucleic acids, to generate a covalently closed circular double stranded nucleic acid molecule comprising the Dual Adapter molecule and target sequence;
d. optionally providing a reaction mixture comprising a library of Dual Adapter molecules of claim 2, a nuclease activity, a DNA polymerase activity, and a DNA ligase activity and a population of target nucleic acids, optionally DNA, cDNA, and/or RNA molecules, wherein the reaction mixture is incubated under conditions that generate single-stranded regions in the target nucleic acid molecules and Dual Adapter molecule HR1 and HR2 regions, wherein the reaction mixture is incubated under conditions that facilitate hybridization of single-stranded complementary regions between the Dual Adapter molecule HR1:HR2 sequences and target nucleic acids, to generate a covalently closed circular double stranded nucleic acid molecule comprising the Dual Adapter molecule and target sequence;
e. providing a reaction mixture comprising a covalently closed circular double stranded nucleic acid molecule comprising the Dual Adapter molecule and target sequence, amplification primers, optionally PCR primers, adapted to hybridize to AS1 and AS2, and reagents to amplify the nucleic acid region bounded by AS1 and AS2 to produce amplification products, optionally adapted for next generation sequencing;
f. optionally providing a reaction mixture comprising a covalently closed circular double stranded nucleic acid molecule comprising the Dual Adapter molecule and target sequence, amplification primers, optionally a primers including a modified nucleotide comprising an affinity tag to facilitate separation, wherein optionally the affinity tag is a biotin molecule or a hapten, adapted to hybridize to AS1 and/or AS2 or other region on the molecule, and reagents to amplify the nucleic acid using rolling circle or isothermal amplification to produce DNA concatemers of repeating units comprising AS1/UMI1/target sequence/UMI2/AS2 that are optionally separated into individual units with a restriction or nicking enzyme or PCR amplified according to step e.