Method and reagent for constructing nucleic acid double-linker single-strand cyclical library
US10479991B2 · kind B2 · utility
Assignee
Inventors
Key dates
| Filing date | Nov 26, 2014 |
| Grant date | Nov 19, 2019 |
| Priority date | — |
| Expiry date | Nov 26, 2034 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC40B50/06
- WIPO fieldBiotechnology
- WIPO sectorChemistry
Abstract
A method and reagent for constructing a nucleic acid double-linker single-strand cyclic library. The method comprises: breaking a nucleic acid into nucleic acid fragments; connecting a first linker sequence; producing by amplification a first product provided with the first linker sequence at either end, where a U nucleobase site is provided on primer sequences and a nicking enzyme recognition sequence is either provided or not provided on same, and a first affinity tag is provided on one of the primer sequences; using USER enzyme to cleave the first product; cyclizing the cleaved first product; treating the cyclization product with either a phosphatase or a nicking enzyme; using a solid-phase vector for combination with a cyclized molecule; performing a restrictive gap translation reaction; removing by digestion any portion that did not undergo the restrictive gap translation reaction; connecting a second linker sequence; producing by amplification a second product provided with the second linker sequence at either end; denaturing the second product, and cyclizing a single-strand nucleic acid molecule. The method allows an increase in the length of library insert fragments, a simp…
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