COMPOSITIONS AND METHODS FOR PRODUCING LIBRARIES WITH CONTROLLED COMPOSITIONS AND SCREENING PROBABILITES

摘要

The invention provides a method for the combinatorial mutagenesis of a parental nucleic acid. The method consists of: (a) extending by enzymatic polymerization a first mutagenic primer annealed to a parental nucleic acid to produce an extension product; (b) treating said extension product with a cleaving reagent selective for a nucleotide sequence present in the parental nucleic acid but absent in the first product; (c) extending by enzymatic polymerization a first PAP annealed to a noncontiguous region of said mutagenic primer to produce a first product having a first mutagenized portion comprising one or more altered nucleotides, the first PAP containing a unique sequence tag associating mutations within the first mutagenic primer with the first PAP; (d) annealing the first product to the parental nucleic acid, and (e) extending by enzymatic polymerization the annealed first product to produce a first modified parental nucleic acid containing a first mutagenized portion. The first product can additionally be amplified. The method also provides the additional step: (f) amplifying the first modified parental nucleic acid containing a first mutagenized portion by polymerase extension of an annealed first SAP to the unique sequence tag contained in the first PAP and an annealed second PAP to the first modified parental nucleic acid, the first and second PAPs corresponding to flanking regions of the parental nucleic acid. The method additionally provides the steps of: (g) repeating steps (a) through (c) one or more times with a second mutagenic primer and a third PAP to noncontiguous regions of the parental nucleic acid to a second product having a second mutagenized portion, the third PAP containing a unique sequence tag associating mutations within the second mutagenic primer with the second PAP, and (h) repeating steps (d) through (e) or steps (d) through (f) one or more times by annealing the second product produced in step (g) to the parental nucleic acid or the first modified parental nucleic acid produced in step (e) or (f) to generate a second modified parental nucleic acid containing a first mutagenized portion and at least one second mutagenized portion. Steps (g) and (h) can be repeated one or more times with tertiary mutagenic primers.