US 12,391,984 B2
Compositions and methods for rolling circle amplification
Rhyan Blaine Dockter, Logan, UT (US)
Filed by 10x Genomics, Inc., Pleasanton, CA (US)
Filed on Aug. 2, 2022, as Appl. No. 17/816,985.
Claims priority of provisional application 63/228,802, filed on Aug. 3, 2021.
Prior Publication US 2023/0044650 A1, Feb. 9, 2023
Int. Cl. C12Q 1/6853 (2018.01); C12Q 1/6841 (2018.01)
CPC C12Q 1/6853 (2013.01) [C12Q 1/6841 (2013.01)] 10 Claims
 
1. A method for analyzing a biological sample, comprising:
(a) hybridizing a circularizable probe or a circularizable probe set to a sequence of a target nucleic acid in the biological sample and ligating the circularizable probe or the circularizable probe set to generate a circularized probe using the target nucleic acid as a template,
wherein the circularized probe comprises a four-hairpin base unit structure comprising a first stem-loop structure, a second stem-loop structure, a third stem-loop structure, and a fourth stem-loop structure, wherein each stem-loop structure independently comprises, from 5′ to 3′, a first stem sequence, a loop region, and a second stem sequence comprising a reverse complement of the first stem sequence,
wherein each stem region of the first stem-loop structure, the second stem-loop structure, the third stem-loop structure, and the fourth stem-loop structure comprises a different sequence,
wherein the loop region of the first stem-loop structure comprises a sequence complementary to the target nucleic acid that is used as the template to generate the circularized probe;
(b) generating a rolling circle-amplification (RCA) product using the circularized probe as a template,
wherein the rolling circle amplification product comprises multiple complementary copies of the four-hairpin base unit structure comprising complementary sequences of the first stem-loop structure, the second stem-loop structure, the third stem-loop structure, and the fourth stem-loop structure; and
(c) detecting the RCA product in the biological sample or in a matrix embedding the biological sample or molecules thereof.