US 12,000,004 B2
Methods for molecularly characterizing cervical cell samples
Jane Shen-Gunther, San Antonio, TX (US)
Assigned to The Government of The United States, as represented by The Secretary of The Army
Filed by The Government of The United States, as represented by The Secretary of The Army, Fort Detrick, MD (US)
Filed on Jun. 14, 2022, as Appl. No. 17/839,803.
Application 17/038,290 is a division of application No. 15/741,891, granted, now 10,829,820, issued on Nov. 10, 2020, previously published as PCT/US2016/049426, filed on Aug. 30, 2016.
Application 17/839,803 is a continuation of application No. 17/038,290, filed on Sep. 30, 2020, granted, now 11,408,039.
Claims priority of provisional application 62/212,555, filed on Aug. 31, 2015.
Prior Publication US 2023/0212682 A1, Jul. 6, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6886 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6883 (2018.01); C12Q 1/70 (2006.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6851 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6883 (2013.01); C12Q 1/708 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/154 (2013.01)] 9 Claims
 
1. A method of determining the methylation level of a plurality of CpG sites of a nucleic acid molecule obtained from a cell of a cervical cell sample, wherein said cell has been infected with a Human papillomavirus having a genotype selected from the group consisting of: 114, 91, 90, 84, 83, 81, 72, 71, 61, 54, 43, 42, 11, 6, 97, 85, 82, 73, 70, 69, 67, 66, 53, 34, 30, 26, a9, 68, 59, 58, 56, 52, 51, 45, 39, 35, 33, 31, 18, and 16 and said nucleic acid molecule has a sequence identity of at least 95% to SEQ ID NO: 3, which comprises
a) converting unmethylated cytosine residues of the nucleic acid molecule to uracil by contacting the nucleic acid molecule with bisulfite to obtain a bisulfite converted nucleic acid molecule;
b) subjecting the bisulfite converted nucleic acid molecule to polymerase chain reaction amplification using a set of primers to obtain amplified nucleic acid molecules; and
c) performing an assay to determine the methylation level of each CpG site in the plurality,
wherein the plurality of CpG sites consist of CpG 1, CpG 2, CpG 3, CpG 4, and CpG 5 of SEQ ID NO: 3.