US 12,286,670 B2
Full-length RNA sequencing
Ivano Legnini, Berlin (DE); Jonathan Alles, Berlin (DE); Nikolaos Karaiskos, Berlin (DE); Salah Ayoub, Berlin (DE); and Nikolaus Rajewsky, Berlin (DE)
Assigned to MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN IN DER, Helmholtz-Gemeinschaft Berlin (DE)
Appl. No. 17/281,269
Filed by MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN IN DER HELMHOLTZ-GEMEINSCHAFT, Berlin (DE)
PCT Filed Aug. 13, 2019, PCT No. PCT/EP2019/071726
§ 371(c)(1), (2) Date Mar. 30, 2021,
PCT Pub. No. WO2020/069791, PCT Pub. Date Apr. 9, 2020.
Claims priority of application No. 18198248 (EP), filed on Oct. 2, 2018.
Prior Publication US 2022/0002797 A1, Jan. 6, 2022
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 1/6806 (2013.01)] 9 Claims
OG exemplary drawing
 
1. A method for analyzing a polyribonucleotide, said method comprising the following steps:
(a) obtaining a labeled polyribonucleotide by linking the 3′ end of said polyribonucleotide and a random sequence of 1 to 20 G and I nucleotide residues; followed by
(b) providing a second molecule comprising:
(b1) a first primer recognition sequence of 18 to 35 nucleotide residues in length that defines the 5′ end of said second molecule, followed by
(b2) a sequence of 5 to 15 C nucleotide residues linked to a sequence of 0 to 12 T nucleotide residues which define the 3′ end of said second molecule;
followed by
(c) obtaining a complex of said labeled polyribonucleotide and said second molecule comprising a double-stranded sequence consisting of sequence (b2) of the second molecule and the complementary 3′ end sequence of the labeled polyribonucleotide; followed by
(d) obtaining an extended second molecule by
(d1) extending the 3′ end of the second molecule comprised in the complex obtained from step (c) by synthesizing a sequence that is complementary to the part of the sequence of the labeled polyribonucleotide which is not part of the double-stranded sequence comprised in the complex; followed by
(d2) extending the 3′ end of the second molecule obtained from step (d1) by adding at least (i) 1 to 5 C nucleotide residues, followed by (ii) a second primer recognition sequence of 18 to 35 nucleotide residues in length,
wherein the polyribonucleotide is an mRNA molecule with a poly(A) tail,
said method further comprising obtaining data for said extended second molecule using a Third Generation sequencing method.