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    • 6. 发明申请
    • METHOD FOR CDNA LIBRARY CONSTRUCTION AND ANALYSIS FROM TRANSFER RNA
    • WO2023020688A1
    • 2023-02-23
    • PCT/EP2021/072902
    • 2021-08-18
    • MAX-PLANCK-GESELLSCHAFT ZUR FÖRDERUNG DER WISSENSCHAFTEN E.V.
    • NEDIALKOVA, Danny D.BEHRENS, AndrewRODSCHINKA, Geraldine
    • C12N15/10
    • The present invention relates to a method for the generation of a cDNA library from transfer RNA (tRNA) comprising (a) optionally ligating at least one DNA adapter to the 3'-end oftRNA, wherein the 3'-end of the DNA adapter is preferably a chain terminator dideoxycytidine, preferably under one or more of the following conditions: (i) crowding reagent at 5% to 35%, preferably 15% to 30%, and most preferably about 25%, (ii) MgCI2 concentration of 1 mM to 15 mM, preferably 3 to 12 mM and most preferably about 10 mM (iii) a temperature of 12°C to 37°C, preferably of about 25°C. (iv) a pH of 6.0 to 9.0, preferably 6.5 to 8.0 and most preferably about 7.0, (v) reducing agent concentration of 0.1 mM to 10 mM, preferably 0.5 to 5 mM and most preferably about 1 mM, and (vi) a reaction time of at least 30 min, preferably at least 1.5 h and most preferably at least 3 h; and (b) reverse transcription in a primer- dependent reaction, in case step (a) is present, or in a template-switching reaction, in case step (a) is absent, of tRNAs into cDNA by a group II intron reverse transcriptase, under the following conditions: (i) KCI or NaCI at a concentration of 20 mM to 250 mM, preferably 50 to 100 mM and most preferably about 75 mM, (ii) MgCh at a concentration of 0.5 mM to 15 mM, preferably 1 to 5 mM and most preferably about 3 mM, (iii) a temperature of 30°C to 65°C, preferably of about 42°C, and (iv) a reaction time of at least 2 h, preferably at least 8 h and most preferably at least 15 h, and preferably (v) a pH of 6.5 to 9.5, preferably 7.0 to 8.5 and most preferably about 8.0, and/or (vi) a reducing agent (DTT) at a concentration of 1 mM to 12.5 mM, preferably 3 to 8 mM and most preferably about 5 mM.