Patent ID: 11964997
Assignee: REALSEQ BIOSCIENCES, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 18:
19. A method for detecting a sample polynucleotide in a sample comprising:
a) ligating a combo adapter to the sample polynucleotide to produce an adapter-polynucleotide ligation product, wherein said combo adapter comprises a 5′-proximal segment, a 3′-proximal segment, and a template-deficient segment linking the single-stranded 5′ proximal segment and the single-stranded 3′ proximal segment that restricts primer extension by a polymerase over the template-deficient segment, and wherein at least one of the 5′ proximal segment or 3′ proximal segment comprises a sequencing adapter;
b) circularizing said adapter-polynucleotide ligation product by ligating its 5′ end to its 3′ end to produce a circularized adapter-polynucleotide ligation product;
c) hybridizing a first primer comprising a sequence at least partially complementary to the 5′-proximal segment of said combo adapter, to said circularized adapter-polynucleotide ligation product;
d) extending said first primer with a polymerase to produce a monomeric nucleic acid that is complementary to the sample polynucleotide flanked by at least a portion of the sequencing adapter, wherein said template-deficient segment allows one cycle of primer extension on said circularized adapter-polynucleotide ligation product, thereby preventing rolling-circle amplification;
e) amplifying said monomeric nucleic acid using said first primer and a second primer, wherein the sequence of the second primer is at least partially complementary to the 3′-proximal segment of said combo adapter; and
f) sequencing the amplified monomeric nucleic acid or portion thereof, thereby detecting the sample polynucleotide.