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الیگونوکلئوتید
Random Mutagenesis with Degenerate Oligonucleotide Primers
The degenerate oligonucleotides are employed as "internal" PCR primers to amplify the left and right portions of the target gene in separate reactions (Fig. 3.47).
Mixing, denaturing, and annealing the left and right fragments produces some DNA molecules that overlap by complementarity and can be extended by DNA polymerase to produce a library of altered genes that have mutated sites in the region of the overlap of the degenerate oligonucleotides.
Two pairs of oligonucleotide primers are used in the PCR; one set of flanking primers anneals to the ends of the target gene (often a cloned gene), and the other set consists of overlapping, internal primers that carry the mutation.
The oligonucleotide prim ers used in the inverse PCR anneal to adjacent sequences in the target gene but are divergently oriented, that is, their 3?
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