US 12,168,799 B2
Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
Clive Gavin Brown, Cambridge (GB); James Clarke, Oxford (GB); Graham Hall, Oxford (GB); Gavin Harper, Sonning (GB); Andrew John Heron, Oxford (GB); and James White, Oxford (GB)
Assigned to Oxford Nanopore Technologies PLC, Oxford (GB)
Filed by Oxford Nanopore Technologies PLC, Oxford (GB)
Filed on Oct. 8, 2021, as Appl. No. 17/496,956.
Application 17/496,956 is a continuation of application No. 16/428,834, filed on May 31, 2019, granted, now 11,168,363.
Application 16/428,834 is a continuation of application No. 15/944,365, filed on Apr. 3, 2018, granted, now 10,597,713.
Application 15/944,365 is a continuation of application No. 14/234,698, granted, now 9,957,560, previously published as PCT/GB2012/051786, filed on Jul. 25, 2012.
Application 17/496,956 is a continuation of application No. 16/782,350, filed on Feb. 5, 2020, granted, now 11,261,487.
Claims priority of provisional application 61/511,436, filed on Jul. 25, 2011.
Prior Publication US 2022/0127669 A1, Apr. 28, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); G01N 27/447 (2006.01); G01N 33/487 (2006.01)
CPC C12Q 1/6869 (2013.01) [G01N 27/44717 (2013.01); G01N 27/44791 (2013.01); G01N 33/48721 (2013.01)] 14 Claims
 
1. A method comprising:
(i) preparing a nucleic acid construct by ligating a polynucleotide bridging moiety to a 3′ terminus of a first strand and a 5′ terminus of a second strand of a double stranded nucleic acid, wherein the first strand of the double stranded nucleic acid comprises a first nucleic acid sequence and the second strand of the double stranded nucleic acid comprises a second nucleic acid sequence that is complementary to the first nucleic acid sequence;
(ii) using a single molecule sequencing apparatus to perform a sequencing reaction that reads through the first strand, the bridging moiety, and the second strand to produce orthogonal proof-reading sequence information for a methylated nucleobase in the first strand of the construct, wherein the movement of the nucleic acid construct through the single molecule sequencing apparatus is controlled by a Phi29 DNA polymerase;
(iii) comparing the sequence information between the first nucleic acid sequence and the second nucleic acid sequence; and
(iv) determining the presence of a methylated nucleobase in the first sequence.