US 12,404,296 B2
Poly(A)-ClickSeq click-chemistry for next generation 3-end sequencing without RNA enrichment or fragmentation
Andrew Routh, Galveston, TX (US); Eric J. Wagner, Galveston, TX (US); Ping Ji, Houston, TX (US); and Elizabeth Jaworski, La Marque, TX (US)
Assigned to Board of Regents, The University of Texas System, Austin, TX (US)
Filed by Board of Regents, The University of Texas System, Austin, TX (US)
Filed on Aug. 25, 2021, as Appl. No. 17/412,024.
Application 17/412,024 is a division of application No. 16/282,159, filed on Feb. 21, 2019, granted, now 11,149,053.
Claims priority of provisional application 62/634,095, filed on Feb. 22, 2018.
Prior Publication US 2022/0002337 A1, Jan. 6, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6869 (2018.01)
CPC C07H 21/04 (2013.01) [C07H 21/02 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6855 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6869 (2013.01); C12Y 207/07006 (2013.01); C12Y 207/07007 (2013.01); C12Y 207/07049 (2013.01)] 13 Claims
 
1. A kit for cDNA synthesis of an RNA 3′ end, a poly (A) junction, and a poly (A) tail of RNA comprising:
one or more vials comprising three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP, dNTPs, and an adaptor sequence-oligo-dT, wherein the terminating modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP are 2′- or 3′-azido-nucleotides: AzGTP, AzCTP and AzATP; or 3′-(O-Propargyl)-NTPs that pair with an alkyne- or azide-modified oligo during a click reaction is a hexanyl-oligo or azide-oligo;
one or more vials comprising a reverse transcriptase;
a cDNA fragment isolating kit;
one or more vials comprising components for chemically ligating a functionalized 5′ adaptor to the cDNA;
a DNA amplification kit for amplifying the chemically-ligated cDNA into an amplification product; and
instructions for amplification of the RNA 3′ end and poly (A) tail junction without fragmentation and enzymatic ligation.