Patent EP-2110435-A1 (EP)
Inventor: VAN DEN BRULLE JAN
IPC: C12N15/10
Filed: 2009-03-31
Abstract: The present invention is related to 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, whereby the method comprises the following steps: 
a) providing a first at least partially double-stranded oligonucleotide having a double-stranded structure which has a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence corresponding to the sequence of the first or third stretch of nucleotides of the elements of the nucleic acid library, whereby the oligonucleotide comprises a recognition site, or part thereof, for a first type IIS restriction enzyme which cuts outside its recognition site, 
b) providing a first oligonucleotide library comprising several members, whereby each member is a second at least partially double-stranded oligonucleotide having a double-stranded structure, whereby the second oligonucleotide comprises a recognition site, or part thereof, for a second type IIS restriction enzyme which cuts outside its recognition site, and a single-stranded overhang, whereby the single-stranded overhang or part thereof is the same for the members of the first oligonucleotide library and whereby such single-stranded overhang is essentially complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide, whereby the double-stranded structure comprises a stretch of nucleotides whereby the members of the first oligonucleotide library differ in the sequence of said stretch of nucleotides and the sequence of the said stretch of nucleotides of the members of the first oligonucleotide library corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library, 
c) combining the first at least partially double-stranded oligonucleotide and the first oligonucleotide library and ligating one molecule of the first at least partially double-stranded oligonucleotide with one molecule of each one member of the first oligonucleotide library via their single-stranded overhangs, whereupon a first ligation product is formed, 
d) cutting the first ligation product with said second type IIS restriction enzyme, whereby the cleavage occurs in the nucleic acid sequence of the member of the first oligonucleotide library, whereby such cleavage provides 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 several members, whereby each member is a third at least partially double-stranded oligonucleotide having a double-stranded structure, whereby the third oligonucleotide comprises a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site, and a single-stranded overhang, whereby the single-stranded overhang or part thereof is different for the members of the second oligonucleotide library and whereby such single-stranded overhangs or part thereof of the third oligonucleotide are essentially complementary to the single-stranded overhangs of the first elongated at least partially double-stranded oligonucleotides, and whereby the members of the second oligonucleotide library comprise a stretch of nucleotides in the double-stranded structure which is identical in all members and corresponds to the sequence, or part thereof, of the third or first stretch of nucleotides of the elements of the nucleic acid library, 
g) combining the plurality of the first elongated at least partially double-stranded oligonucleotides and the second oligonucleotide library and ligating each one molecule of the plurality of the first elongated at least partially double-stranded oligonucleotides with one molecule of each of the members of the second oligonucleotide library, whereby the single-stranded overhang of the first elongated at least partially double-stranded oligonucleotide is essentially complementary to the overhang of the third at least partially double-stranded oligonucleotide, whereupon a second ligation product is formed, 
h) cutting the second ligation product with the third type II S restriction enzyme, whereby the cleavage occurs in the nucleic acid sequence of the third at least partially double-stranded oligonucleotides, 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 a fourth at least partially double-stranded oligonucleotide having a double-stranded structure which has a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence essentially complementary to the single-stranded overhang of the second elongated at least partially double-stranded oligonucleotide, whereby the oligonucleotide comprises a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site, 
k) combining the fourth at least partially double-stranded oligonucleotide and the plurality of the second elongated at least partially double-stranded oligonucleotides and ligating one molecule of the fourth oligonucleotide and each one molecule of the plurality of the second elongated at least partially double-stranded oligonucleotides forming a third ligation product; 
l) cutting the third ligation product with the fourth type IIS restriction enzyme, whereby the cleavage occurs in the nucleic acid sequence of the fourth at least partially double-stranded oligonucleotide(s), providing for a plurality of third elongated at least partially double-stranded oligonucleotides and shortened fourth at least partially double-stranded oligonucleotide, and 
m) removing the shortened fourth at least partially double-stranded oligonucleotide.