A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications reduced the fidelity of Taq polymerase during DNA synthesis without significantly decreasing PCR amplification. The resulting PCR products could be cloned to produce random mutant libraries or transcribed directly if a T7 promoter was incorporated into the primers. This method was used to mutagenize the gene encoding the Tetrahymena ribozyme with a mutation rate of 0.66% ± 0.13% per position per PCR, as determined by sequence analysis. Mutations were evenly distributed and followed a Poisson distribution. In vitro mutagenesis allows probing of structurally and functionally important regions in cloned genes. Random mutagenesis combined with screening is useful when functionally significant positions are unknown. Several methods for introducing random mutations include chemical mutagenesis, nucleotide analogs, bacterial passage, randomized oligonucleotides, and inaccurate polymerase copying. Inaccurate polymerase copying is preferred due to its simplicity and versatility. The fidelity of various DNA polymerases was measured, including Taq, which had the lowest fidelity. When combined with PCR's amplification power, Taq was the best candidate for in vitro mutagenesis. Its error rate was 0.001–0.02% per nucleotide per pass, but this was insufficient for mutagenizing a gene. Modifications to reaction conditions or polymerase were needed to reduce fidelity. A previous study described a random mutagenesis method based on PCR modifications, achieving a 2% mutation rate per position per PCR. However, this method had a strong GC bias. The authors developed a new PCR mutagenesis protocol with no mutational bias and an overall error rate of 0.66% ± 0.13% per position per PCR. The study tested various PCR conditions and found that the preferred condition resulted in no strong mutational bias. The mutation rate was 0.66% ± 0.13% per position per PCR, with no significant differences between independent experiments. The mutations were randomly distributed, with no clustering or hotspots. The DNA yield in mutagenic PCR was similar to standard PCR. The study highlights the importance of maximizing PCR fidelity for preparative purposes but also notes the usefulness of low-fidelity polymerization when mutational bias is not desired. PCR mutagenesis can be used to randomize any cloned gene for generating mutant libraries. The method described here provides a way to randomize genes without sequence bias, making it suitable for generating mutant libraries that can be screened for desired phenotypes. The study also discusses the potential for modifying DNA polymerases to achieve low-fidelity enzymes.A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications reduced the fidelity of Taq polymerase during DNA synthesis without significantly decreasing PCR amplification. The resulting PCR products could be cloned to produce random mutant libraries or transcribed directly if a T7 promoter was incorporated into the primers. This method was used to mutagenize the gene encoding the Tetrahymena ribozyme with a mutation rate of 0.66% ± 0.13% per position per PCR, as determined by sequence analysis. Mutations were evenly distributed and followed a Poisson distribution. In vitro mutagenesis allows probing of structurally and functionally important regions in cloned genes. Random mutagenesis combined with screening is useful when functionally significant positions are unknown. Several methods for introducing random mutations include chemical mutagenesis, nucleotide analogs, bacterial passage, randomized oligonucleotides, and inaccurate polymerase copying. Inaccurate polymerase copying is preferred due to its simplicity and versatility. The fidelity of various DNA polymerases was measured, including Taq, which had the lowest fidelity. When combined with PCR's amplification power, Taq was the best candidate for in vitro mutagenesis. Its error rate was 0.001–0.02% per nucleotide per pass, but this was insufficient for mutagenizing a gene. Modifications to reaction conditions or polymerase were needed to reduce fidelity. A previous study described a random mutagenesis method based on PCR modifications, achieving a 2% mutation rate per position per PCR. However, this method had a strong GC bias. The authors developed a new PCR mutagenesis protocol with no mutational bias and an overall error rate of 0.66% ± 0.13% per position per PCR. The study tested various PCR conditions and found that the preferred condition resulted in no strong mutational bias. The mutation rate was 0.66% ± 0.13% per position per PCR, with no significant differences between independent experiments. The mutations were randomly distributed, with no clustering or hotspots. The DNA yield in mutagenic PCR was similar to standard PCR. The study highlights the importance of maximizing PCR fidelity for preparative purposes but also notes the usefulness of low-fidelity polymerization when mutational bias is not desired. PCR mutagenesis can be used to randomize any cloned gene for generating mutant libraries. The method described here provides a way to randomize genes without sequence bias, making it suitable for generating mutant libraries that can be screened for desired phenotypes. The study also discusses the potential for modifying DNA polymerases to achieve low-fidelity enzymes.