Mutagenesis methods and compositions

A method for introducing a plurality of mutations to a nucleic acid construct comprising a target sequence, a first marker sequence encoding a first protein having an inoperable first marker activity, and a second marker sequence encoding a second protein having an operable second marker activity, said method comprising the steps of:

• (a) annealing to a single-stranded form of said nucleic acid construct
• a first oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said first oligonucleotide is selected so as to change a nucleotide at a first position of said nucleic acid construct where said first position is within said target sequence, and to introduce or remove a first restriction site at a second position of said nucleic acid construct;
• a second oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said second oligonucleotide is selected so as to change one or more nucleotides in said first marker sequence to produce a mutated first marker sequence in a manner which results in an operable first marker activity;
• and
• a third oligonucleotide having a sequence substantially complementary to a portion of said nucleic acid construct, wherein the sequence of said third oligonucleotide is selected so as to change one or more nucleotides in said second marker sequence to produce a mutated second marker sequence in a manner which results in an inoperable second marker activity, thereby forming a primary annealed product;
• (b) transforming a host cell with said primary annealed product;
• (c) screening or selecting progeny of transformed host cells having said operable first marker activity;
• (d) identifying progeny screened or selected and containing a mutated nucleic acid construct containing a mutated target sequence and having said introduced or removed first restriction site;
• (e) annealing to a single-stranded form of said mutated nucleic acid construct
• a fourth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said fourth oligonucleotide is selected so as to change a nucleotide at a first position of said mutated nucleic acid construct where said first position is within said mutated target sequence, and to introduce or remove a second restriction site at a second position of said mutated nucleic acid construct;
• a fifth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said fifth oligonucleotide is selected so as to restore said first marker sequence in a manner which results in said inoperable first marker activity;
• and
• a sixth oligonucleotide having a sequence substantially complementary to a portion of said mutated nucleic acid construct, wherein the sequence of said sixth oligonucleotide is selected so as to restore said second marker sequence in a manner which results in said operable second marker activity, thereby forming a secondary annealed product;
• (f) transforming a host cell with said secondary annealed product;
• (g) screening or selecting progeny of transformed host cells having said operable second marker activity;
• and
• (h) identifying progeny screened or selected and containing a further mutated nucleic acid construct having said introduced or removed second restriction site.