Poly(A)-ClickSeq click-chemistry for next generation 3-end sequencing without RNA enrichment or fragmentation
US11149053B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Feb 21, 2019 |
| Grant date | Oct 19, 2021 |
| Priority date | — |
| Expiry date | Nov 29, 2039 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12Y207/07049
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
The present invention includes a method and kit for cDNA synthesis of a 3′UTR/poly(A) tail junction of cellular RNA comprising: obtaining RNA comprising a 3′UTR/poly(A) junction and a poly(a) tail; combining the RNA with three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP, dNTPs, and adaptor sequence-oligo-dT; performing reverse transcription of the RNA with a reverse transcriptase primed with the adaptor sequence-oligo-dT to form terminated cDNA fragments that are stochastically terminated upstream of the 3′UTR/poly(A) junction, but not within the poly(A) tail; isolating the terminated cDNA fragments; chemically ligating a functionalized 5′ adaptor to the terminated cDNA; and amplifying the chemically-ligated cDNA into an amplification product, wherein the cDNA is enriched for sequences at the 3′UTR/poly(A) tail junction without fragmentation or enzymatic ligation.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.