Method for analyzing impurities of oligonucleotide sequence based on high-throughput sequencing and application
An oligonucleotide, high-throughput technology, applied in biochemical equipment and methods, microbial measurement/testing, library creation, etc.
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[0063] Example 1. A method for analyzing oligonucleotide sequence impurities based on high-throughput sequencing
[0064] 1. Oligonucleotide plus poly tail
[0065] 1. Add a poly A tail to the 3' end of the oligonucleotide sequence, and prepare a tailing reaction system according to the reagents and dosage in Table 1. The oligonucleotide sequence was as follows: 5'-CAGAGCAGCTTGTCTTTTCTTC-3' (SEQ ID NO: 5). Oligonucleotide sequences were synthesized by Shanghai Jierui Bioengineering Co., Ltd.
[0066] Table 1 is the tailing reaction system
[0067] Reagent
Amount added (μL)
13
Oligonucleotides (100 μM)
1
dATP (25μM)
0.5
5×TdT Buffer
4
terminal transferase
1.5
total capacity
20 μL
[0068] 2. React at 37°C for 25 minutes.
[0069] 3. React at 70°C for 10 minutes to inactivate terminal transferase.
[0070] 2. Reverse extension and amplification
[0071] 1. Add the reagents sh...
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