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. 2006 Nov 16;34(21):e147. doi: 10.1093/nar/gkl896

Figure 4.

Figure 4

Oligonucleotide-mediated codon substitution in Rb. (a) Single-stranded DNA oligonucleotide Rb-CTTT was designed to replace an asparagine by a phenylalanine at position 750 in exon 22 of the Rb gene. Mismatching bases are indicated in red. Arrows indicate the location of PCR primers. (b) Primer pair 1/2 was used to amplify a 738 bp fragment from pools of cells. This fragment was used in a second PCR round using the nested primer pairs 3/4 or 5/6 of which primers 3 and 6 are specific for the CTTT mutation. (c) Sequence analysis of Rb mRNA in a purified mutant ES cell clone revealed the presence of the CTTT mutation, replacing the asparagine at position 750 by a phenylalanine. (d) PCR-based detection of RbN750F mutation in genomic DNA. PCRs were conducted with primer pairs 1/2 and 3/4, yielding a 213-bp product specific for the CTTT mutation. Lane M, molecular mass standards; lane 1, Rb+/N750F ES cell clone; lane 2, Rb+/N750F mouse; lane 3, wild-type littermate; lane 4, water control; * indicates non-specific band.