METHOD OF CONSTRUCTING NUCLEIC ACID LIBRARY

摘要

It is intended to establish a method of constructing a nucleic acid library by using the Y-ligation method. Namely, a method of constructing a nucleic acid library which involves the following steps (1) to (7): (1) preparing a first single-stranded nucleic acid and a second single-stranded nucleic acid each having a stem sequence and a branch sequence and a base sequence encoding an amino acid at the 3'-end; (2) hybridizing the first single-stranded nucleic acid with the second single-stranded nucleic acid between the stem sequences thereof; (3) treating the hybridized product with T4 RNA ligase to thereby ligate the 3'-end of the single-stranded nucleic acid to the 5'-end of the second single-stranded nucleic acid; (4) using the double-stranded nucleic acid obtained in the step (3) as a template, carrying out PCR with the use of primers modified in the 5'-side with a substance having affinity to thereby prepare double-stranded nucleic acids; (5) treating two double-stranded nucleic acids obtained in the step (4) with a restriction enzyme to give double-stranded nucleic acids respectively having base sequences encoding the first and second amino acids at the 3'- or 5'-end; (6) using the binding ability of the substance having affinity introduced in the step (4), preparing single-stranded nucleic acids from the double-stranded nucleic acids obtained in the step (5); and (7) using the single-stranded nucleic acids obtained in the step (6), repeating the steps (2) to (6) needed times.