Patent ID: 11859246
Assignee: ACCURAGEN HOLDINGS LIMITED
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for processing or analyzing a biological sample of a subject, comprising:
(a) obtaining said biological sample of said subject;
(b) using a nucleic acid molecule derived from said biological sample to generate a circular nucleic acid molecule; and
(c) subjecting said circular nucleic acid molecule or derivative thereof to amplification in a reaction mixture comprising a first primer, a second primer, a third primer, and said circular polynucleotide, said amplification comprising:
(i) hybridizing said first primer to a first sequence of said circular polynucleotide, the first primer comprising a first 3′ end that specifically hybridizes to the target polynucleotide via sequence complementarity and a first 5′ end comprising a first common sequence that does not specifically hybridize to the target polynucleotide via sequence complementarity;
(ii) extending said first primer to generate a first concatemer comprising a first single-stranded polynucleotide hybridized to said circular polynucleotide, wherein said first single-stranded polynucleotide comprises a second sequence that is complementary to said first sequence of said circular polynucleotide;
(iii) separating said first single-stranded polynucleotide from said circular polynucleotide (iv) hybridizing said second primer to a portion of said second sequence of said first single-stranded polynucleotide of (iii) and said third primer to a third sequence of said circular polynucleotide, the third primer comprising a second 3′ end that specifically hybridizes to the circular polynucleotide via sequence complementarity and a second 5′ end comprising a second common sequence that does not specifically hybridize to the circular polynucleotide via sequence complementarity, the second primer comprises a sequence that specifically hybridizes to the first common sequence and/or the second common sequence via sequence complementarity;
(v) extending said second primer to generate a nucleic acid molecule comprising said first single-stranded polynucleotide and a complement of said first single-stranded polynucleotide, and extending said third primer to generate a second concatemer comprising a second single-stranded polynucleotide hybridized to said circular polynucleotide; and
(vi) amplifying said nucleic acid molecule of (v) to generate a plurality of amplicons, and

(d) subjecting said plurality of amplicons or derivative thereof to sequencing to identify a sequence of said nucleic acid molecule derived from said biological sample.