A method is provided for producing a library of mutagenized
polynucleotides from a target sequence comprising (a) taking a sample
comprising: (i) a target sequence including a section to be mutagenized,
(ii) a library of first primers where the first primers include a first
fixed sequence and a first unknown sequence 3' to the first fixed
sequence, the first unknown sequence varying within the library of first
primers, and (iii) a library of second primers where the second primer
include a second fixed sequence that differs from the first fixed
sequence, and a second unknown sequence 3' to the second fixed sequence,
the second unknown sequence varying within the library of second primers;
(b) performing one or more cycles of primer extension amplification on
the sample in the presence of at least one polymerase such that a member
of the library of the first primers is extended relative to the target
sequence; and (c) performing one or more additional cycles of primer
extension amplification on the sample such that a member of the library
of the second primers is extended relative to the first primer that was
extended in step (b) to form the library of mutagenized polynucleotides.
The mutagenesis produces a library of mutagenized targeted sequences with
random truncations.