Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides |
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Authors: | Kenner Oliver Lutomska Anna Speit Günter Vogel Walther Kaufmann Dieter |
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Affiliation: | Department of Human Genetics, University of Ulm, D 89070 Ulm, Germany. |
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Abstract: | The repair of point mutations in hprt gene by single-stranded oligonucleotides represents a model to test targeted nucleotide exchange. We studied the concurrent nucleotide exchange of two or three nucleotides in the hprt deficient hamster cell line V79-151. The used oligonucleotides resulted in mismatches at two (151, 159) or three (151, 144, and 159) hprt positions. The hprt point mutation at position 151 was repaired in about 2/10(6) cells as shown by hprt sequencing in clones surviving HAT selection. The second nucleotide exchange at hprt position 159 was found in 7% of these HAT selected clones. Using oligonucleotides resulting in three mismatches, 29% of the clones showed nucleotide exchanges at the two hprt positions (151, 144) and about 4% at three positions (151, 144, and 159). These results indicate that single-stranded oligonucleotides can generate two or three nucleotide exchanges in a mammalian chromosomal gene. |
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Keywords: | Targeted nucleotide exchange Single-stranded oligonucleotides Point mutation hprt V79 |
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