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Mutagenesis reactions. The mutagenesis protocol and solutions derive
from the Bio-Rad Muta-Gene Mutagenesis Kit, version 2. Six mutants
were generated containing ''silent'' base substitutions that reduced
the local A-bias in a short segment of pol without altering the amino
acid sequence using the following, partially overlapping 5' phosphorylated
oligos (numbers in parentheses indicate 5' coordinate): H2 (2712)
\dna{5'-CCATACAACACCCCCGTGTTCGCCATCAAGAAGAAGGACAGTACCAAATGGAG-3'},
H3 (2748) \dna{5'-GACAGTACCAAGTGGCGCAAGCTGGTGGACTTCAGGAGCTGAACAAGAGGACCCACTGG-3'},
H4 (2808) \dna{5'-TGGGAAGTGCAGCTGGGCATCCCCCACCCCGCCGGGTTAAAGCAGAAGAAGTCCGTAACAGTACTG-3'},
H5 (2865) \dna{5'-GTAACAGTGCTGGATGTGGGCGATGCCTACTTCTCCGTGCCCCTGGATAAAGAC-3'},
H6 (2922) \dna{5'-AGGAAGTACACCGCCTTCACCATCCCCAGTATCAACAACGAGACCCCAGGG-3'},
and H7 (2967) \dna{5'-CCAGGGATCCGCTACCAAGGGCTCCCCCGCAATATTC-3'}.
Mutagene kit annealing buffer was added to 0.6 pmol ssDNA template
to a final volume of 10 ul. Reactions were heated in a heating block
to 94° and then cooled at a rate of 2 degrees/minute until 40° was
reached, then they were
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