Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides.
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.
Kenner
Oliver
O
Department of Human Genetics, University of Ulm, D 89070 Ulm, Germany.
Lutomska
Anna
A
Speit
Günter
G
Vogel
Walther
W
Kaufmann
Dieter
D
D000595:Amino Acid Sequence; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D005821:Genetic Techniques; D007041:Hypoxanthine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics*; D008957:Models, Genetic; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017354:Point Mutation
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D007041:Hypoxanthine Phosphoribosyltransferase