A PCR-based strategy for extensive mutagenesis of a target DNA sequence.
A mixed population of mutagenic oligonucleotide primers was used to generate a set of point mutations in a short region of a retroviral gene by PCR amplification. The mixed population of mutagenic primers was generated by incorporating a mixture of A, G, C and T at specific sites during oligonucleotide synthesis. With the proportions of mutagenic nucleotides used for our experiments, 47 percent of the 213 clones analyzed had one or more point mutation in the target DNA sequence. In addition, unpredicted mutations were observed that contributed to the mutagenic complexity of the population. We have found this approach to be an efficient means for extensive mutagenesis of a defined target DNA sequence.
Morrison
H G
HG
New England Regional Primate Research Center, Harvard Medical School.
Desrosiers
R C
RC
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D015686:Gene Products, env / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D014760:Viral Fusion Proteins
D004279:DNA, Viral; D015686:Gene Products, env; D015682:Retroviridae Proteins, Oncogenic; D014760:Viral Fusion Proteins; C059580:transmembrane protein, Simian immunodeficiency virus; D004247:DNA