Process for sequence saturation mutagenesis (SeSaM)

1. A process for the mutagenesis of a double-stranded polynucleotide sequence (master sequence) of n base-pairs having a (+)-strand and a complementary (−)-strand comprising the steps

(i) creating a collection of single-stranded fragments of the (+)-strand of the master sequence wherein all members of the collection have the same 5′-terminus and have a deletion in the 3′-terminus such that the collection represents (+)-strands with a length of n−1, n−2, n−3, . . . nucleotides;

(ii) introducing at least one universal or degenerate nucleotide at the 3′-terminus of the (+)-strands produced in step (i);

(iii) elongating the (+)-strands produced in step (ii) to the full length of the master sequence using the (−)-strand or fragments thereof of the master sequence as a template strand for the elongation; and

(iv) synthesizing a (−)-strand by using the (+)-strand produced in step (iii) as a template strand thereby effecting mutations in the (−)-strand at the positions of the previous universal or degenerate nucleotides compared to the master sequence.

2. The process of claim 1 , wherein the collection of single-stranded fragments in step (i) is created by incorporating nucleotide analogs and subsequent cleavage in alkaline or acidic solution.

3. The process of claim 2 , wherein the nucleotide analog is an alpha-phosphothioate nucleotide and oxidative cleavage is achieved by iodine at the phosphothioate bonds.

4. The process of claim 1 , wherein step (ii) comprises elongating the collection of single-stranded fragments produced in step (i) with at least one universal or degenerate nucleotide by enzymatic or chemical methods.

5. The process of claim 4 , wherein terminal deoxynucleotidyl transferases or DNA polymerases or DNA/RNA ligases are used for elongation.

6. The process of claim 1 , wherein deoxyinosine, 3-nitropyrrole, 5-nitroindole or a nucleotide analog with promiscuous base pairing property is used as a universal nucleotide in step (ii).

7. The process of claim 1 , wherein N 6 -methoxy-2,6-diaminopurine (K), N 6 -methoxy-aminopurine (Z), hydroxylaminopurine (HAP), 2′-deoxyribonucleoside triphosphate (dyTP), 6H,8H-3,4-dihydropyrimidol [4,5-c][1,2] oxazin-7-one (P), N 4 -aminocytidine, N 4 -hydroxy-2′-deoxycytidine, N 4 -methoxy-2′-deoxycytidine, 8-oxodeoxy-guanosine triphosphate (8-oxo-G) or a nucleotide analog with promiscuous base pairing property is used as degenerate nucleotide in step (ii).

8. The process of claim 1 , wherein an oligonucleotide of the general formula

p(U) n (N) b *(S) c [TERM]

with

p=5′-phosphate or hydroxy-group or any chemical group capable of forming diester bonds

U=universal or degenerate nucleotides

a=arbitrary integral number from 0 to 10000

N=mixture of four bases (A/T/G/C (standard nucleotides))

b=arbitrary integral number from 0 to 100

*=cleavable group such as phosphothioate bonds in phosphothioate nucleotides

S=standard nucleotide or nucleotide analog

c=arbitrary integral number from 0 to 100

[TERM]=a dye terminator or any group preventing elongation of the oligonucleotide, with the proviso that a+b>0,

is used in step (ii) to introduce the at least one universal or degenerate nucleotide to the collection of single-stranded fragments created in step (i).

9. The process of claim 8 , wherein the oligonucleotide is designed in a way that

(a) stop codons and/or

(b) amino acids which disrupt secondary structures,

are avoided in the collection of the mutagenized polynucleotide sequences.

10. The process of claim 8 , wherein the oligonucleotide is designed in a way that

(a) transition mutations or

(b) transversion mutations,

are effected in the collection of the mutagenized polynucleotide sequences.

11. The process of claim 8 , wherein a DNA/RNA ligase is used for ligation of the oligonucleotides to the single-stranded fragments created in step (i), and wherein single-stranded fragments created in step (i) which are not ligated to the oligonucleotide are removed using an exonuclease.

12. The process of claim 1 , wherein the elongation in step (iii) is effected by a PCR reaction.

13. The process of claim 1 , wherein step (iii) comprises synthesizing a (−)-single stranded plasmid polynucleotide sequence from a double-stranded plasmid harboring the master sequence using a primer which anneals downstream of the (+)-strand of the master sequence, and annealing the (−)-single stranded-plasmid polynucleotide sequence with the (+)-strand produced in step (ii), and elongating the (+)-strand.

14. The process of claim 1 , wherein step (iii) comprises synthesizing a (−)-single-stranded plasmid harboring the master sequence using a primer which anneals downstream of the (+)-strand of the master sequence in the presence of uracil and standard nucleotides and after elongating the (+)-strand produced in step (ii), digesting the uracil carrying (−)-single-stranded plasmid with uracil glycosylase.

15. The process of claim 1 , wherein a PCR amplification is used after step (iii) in order to synthesize a (−)-strand complementary to the (+)-strand produced in step (iii), thereby effecting a double-stranded master sequence carrying mutations.