| 摘要 |
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) creation of 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) introduction of at least one universal or degenerate nucleotide at the 3′-terminus of the (+) strand produced in step (i); (iii) elongation of the (+)-strand produced in step (ii) to the full length of the master sequence using the (−)-strand or fragments thereof as a template strand for the elongation; (iv) synthesis of 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.
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