Patent ID: 9115352
Filing Date: 2015-08-25
Classification: C12N

Abstract:
1. A method for preparing a nucleic acid library comprising a plurality of elements, whereby each element of said nucleic acid library comprises a first stretch of nucleotides, a second stretch of nucleotides and a third stretch of nucleotides, whereby the sequence of the first stretch of nucleotides and of the third stretch of nucleotides are identical in each element of the nucleic acid library and the elements of the nucleic acid library differ in the sequence of the second stretch of nucleotides, the method comprising the following steps: a) providing a first at least partially double-stranded oligonucleotide comprising b) providing a first oligonucleotide library comprising a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprising c) ligating the first at least partially double-stranded oligonucleotide with the members of the first oligonucleotide library via their single-stranded overhangs, whereupon first ligation products are formed; d) cutting the first ligation products with said second type IIS restriction enzyme in the nucleic acid sequence of the members of the first oligonucleotide library, and providing for a plurality of first elongated at least partially double-stranded oligonucleotides and for shortened second at least partially double-stranded oligonucleotides; e) removing the shortened second at least partially double-stranded oligonucleotides; f) providing a second oligonucleotide library comprising a plurality of members, whereby each member is a third at least partially double-stranded oligonucleotide comprising g) ligating the plurality of first elongated at least partially double-stranded oligonucleotides with the members of the second oligonucleotide library, whereby the single-stranded overhang of the first elongated at least partially double-stranded oligonucleotide is complementary to the overhang of the third at least partially double-stranded oligonucleotides, whereupon second ligation products are formed; h) cutting the second ligation products with the third type IIS restriction enzyme in the nucleic acid sequence of the third at least partially double-stranded oligonucleotides, and providing for a plurality of second elongated at least partially double-stranded oligonucleotides and shortened third at least partially double-stranded oligonucleotides; i) removing the shortened third at least partially double-stranded oligonucleotides; j) providing fourth at least partially double-stranded oligonucleotides comprising k) ligating the fourth oligonucleotides and the plurality of the second elongated at least partially double-stranded oligonucleotides to form third ligation products; l) cutting the third ligation products with the fourth type IIS restriction enzyme in the nucleic acid sequence of the fourth at least partially double-stranded oligonucleotides, and providing for a plurality of third elongated at least partially double-stranded oligonucleotides and shortened fourth at least partially double-stranded oligonucleotides; and m) removing the shortened fourth at least partially double-stranded oligonucleotides; and n) obtaining the nucleic acid library