Method for the preparation of a nucleic acid library
First Claim
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 comprisingaa) 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; and
ab) 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 a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprisingba) a recognition site, or part thereof, for a second type IIS restriction enzyme which cuts outside its recognition site;
bb) a single-stranded overhang, whereby the single-stranded overhang is the same for each of the members of the first oligonucleotide library and is complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide; and
bc) whereby the members of the first oligonucleotide library differ in the sequence of a stretch of nucleotides and the sequence of said stretch of nucleotides corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library, wherein one or more nucleotide positions within the stretch of nucleotides differs at a controlled frequency;
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 comprisingfa) a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site;
fb) a single-stranded overhang, whereby the single-stranded overhang is different for the members of the second oligonucleotide library and whereby such single-stranded overhangs of the third oligonucleotide are complementary to the single-stranded overhangs of the first elongated at least partially double-stranded oligonucleotides; and
fc) a stretch of nucleotides 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) 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 comprisingja) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhang of the second elongated at least partially double-stranded oligonucleotide; and
jb) a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site;
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 librarywherein the nucleic acid library is a library of nucleic acid molecules coding for a peptide, polypeptide, protein, or functional nucleic acid;
wherein the peptide, polypeptide, or protein comprises one or several constant regions, and one or several variable regions;
wherein the one or several of the variable regions is/are encoded by the sequence of the second stretch of nucleotides of the elements of the nucleic acid library; and
wherein the second stretch of nucleotides of the elements of the nucleic acid library is the only difference between said elements.
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Abstract
The present invention is related to a method for preparing a nucleic acid library comprising a plurality of various elements or nucleic acid molecules that differ in a controlled manner at one or several distinct nucleotide positions.
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Citations
21 Claims
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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:
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a) providing a first at least partially double-stranded oligonucleotide comprising aa) 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; and ab) 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 a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprising ba) a recognition site, or part thereof, for a second type IIS restriction enzyme which cuts outside its recognition site; bb) a single-stranded overhang, whereby the single-stranded overhang is the same for each of the members of the first oligonucleotide library and is complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide; and bc) whereby the members of the first oligonucleotide library differ in the sequence of a stretch of nucleotides and the sequence of said stretch of nucleotides corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library, wherein one or more nucleotide positions within the stretch of nucleotides differs at a controlled frequency; 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 fa) a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site; fb) a single-stranded overhang, whereby the single-stranded overhang is different for the members of the second oligonucleotide library and whereby such single-stranded overhangs of the third oligonucleotide are complementary to the single-stranded overhangs of the first elongated at least partially double-stranded oligonucleotides; and fc) a stretch of nucleotides 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) 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 ja) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhang of the second elongated at least partially double-stranded oligonucleotide; and jb) a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site; 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 wherein the nucleic acid library is a library of nucleic acid molecules coding for a peptide, polypeptide, protein, or functional nucleic acid; wherein the peptide, polypeptide, or protein comprises one or several constant regions, and one or several variable regions; wherein the one or several of the variable regions is/are encoded by the sequence of the second stretch of nucleotides of the elements of the nucleic acid library; and wherein the second stretch of nucleotides of the elements of the nucleic acid library is the only difference between said elements. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 20, 21)
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15. 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:
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a) providing a first at least partially double-stranded oligonucleotide comprising aa) 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; and ab) 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 a plurality of members, whereby each member is a second at least partially double-stranded oligonucleotide comprising ba) 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 is the same for each of the members of the first oligonucleotide library and is complementary to the single-stranded overhang of the first at least partially double-stranded oligonucleotide; and bb) 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, wherein one or more nucleotide positions within the stretch of nucleotides differs at a controlled frequency; c) ligating the first at least partially double-stranded oligonucleotide with 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 member 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, whereby such plurality of first elongated at least partially double-stranded oligonucleotides consist of various molecule species which differ in the sequence of the single-stranded overhang; e) removing the shortened second at least partially double-stranded oligonucleotides; f) providing a second oligonucleotide library comprising several subgroups of oligonucleotides and each subgroup comprising several members, whereby each member is a third at least partially double-stranded oligonucleotide comprising fa) a recognition site, or part thereof, for a third type IIS restriction enzyme which cuts outside its recognition site; fb) a single-stranded overhang, whereby the single-stranded overhang is different for the various subgroups of oligonucleotides of the second oligonucleotide library and whereby the single-stranded overhang of each one subgroup of the second oligonucleotide library is complementary to the single-stranded overhang of each of the first elongated at least partially double-stranded oligonucleotides; and fc) a stretch of nucleotides which is different in the members of each subgroup of the second oligonucleotide library and corresponds to the sequence, or part thereof, of the second stretch of nucleotides of the elements of the nucleic acid library; g) ligating the plurality of the first elongated at least partially double-stranded oligonucleotides with the members of the second oligonucleotide library, whereby those molecules are ligated at the overhangs of which are complementary to each other, whereupon second ligation products are formed; h) cutting the second ligation products with the third type II S restriction enzyme in the nucleic acid sequence of the third at least partially double-stranded oligonucleotide, and providing for a plurality of second elongated at least partially double-stranded oligonucleotides and shortened third at least partially double-stranded oligonucleotides, whereby such plurality of second elongated at least partially double-stranded oligonucleotides consists of various subgroups which differ in their single-stranded overhangs; i) removing the shortened third at least partially double-stranded oligonucleotides; j) providing a third oligonucleotide library comprising several members, whereby each member is a fourth at least partially double-stranded oligonucleotide comprising ja) a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhangs of the second elongated at least partially double-stranded oligonucleotides; and jb) a stretch, preferably adjacent to said overhang which is identical in the various members and provides for the third or first stretch of nucleotides of the elements of the nucleic acid library, whereby the members of the third oligonucleotide library differ in their single-stranded overhangs and the overhangs correspond to the second stretch, or part thereof, of the nucleic acid library; and jc) a recognition site, or part thereof, for a fourth type IIS restriction enzyme which cuts outside its recognition site; k) combining the plurality of second elongated at least partially double-stranded oligonucleotides and the third oligonucleotide library and ligating each molecule of the members of the third oligonucleotide library with one molecule of the plurality of the second elongated at least partially double-stranded oligonucleotides, whereby those molecules are ligated and the overhangs of which are complementary to each other, whereupon third ligation products are formed; l) cutting the third ligation product with the fourth type IIS restriction enzyme in the nucleic acid sequence of the member of the third oligonucleotide library, providing for a plurality of third elongated at least partially double-stranded oligonucleotides and shortened fourth at least partially double-stranded oligonucleotides; m) optionally removing the shortened fourth at least partially double-stranded oligonucleotides; n) providing a fifth at least partially double-stranded oligonucleotide which has a single-stranded overhang, whereby the single-stranded overhang provides for a nucleotide sequence complementary to the single-stranded overhang of the plurality of third elongated at least partially double-stranded oligonucleotides, whereby the fifth oligonucleotide comprises a recognition site, or part thereof, for a fifth type IIS restriction enzyme which cuts outside its recognition site; o) combining the plurality of third elongated at least partially double-stranded oligonucleotides and the fourth oligonucleotide library and ligating each molecule of the plurality of the third elongated at least partially double-stranded oligonucleotides with one molecule of the fifth at least double-stranded oligonucleotide, whereupon a fourth ligation product is formed; p) cutting the fourth ligation product with the fifth type IIS restriction enzyme in the nucleic acid sequence of the fifth at least partially double-stranded oligonucleotide, providing for a plurality of fourth elongated at least partially double-stranded oligonucleotides and the shortened fifth at least partially double-stranded oligonucleotides; q) optionally removing the shortened fifth at least partially double-stranded oligonucleotides; r) obtaining the nucleic acid library, wherein the nucleic acid library is a library of nucleic acid molecules coding for a peptide, polypeptide, or protein; whereby the peptide, polypeptide, or protein comprises one or several constant regions, and one or several variable regions; wherein the one or several of the variable regions is/are encoded by the sequence of the second stretch of nucleotides of the elements of the nucleic acid library; and wherein the second stretch of nucleotides of the elements of the nucleic acid library is the only difference between said elements. - View Dependent Claims (16, 17)
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Specification