Aging Aging

HPRT Mutational Spectrum 195Table 2HPRT Exon-Specific Primers Used for Genomic PCR and Sequencing (8)ExonPrimer sequence1 E1F b : (1599) 5'-GCG CCT CCG CCT CCT CCT CTG-3' (1619)(65°C) a E1R c : (1801) 5'-CCG CCC GAG CCC GCA CTG-3' (1784)2 E2F c : (14678) 5'-TGG GAT TAC ACG TGT GAA CCA ACC-3' (14701)(61°C) E2R b : (15249) 5'-GAC TCT GGC TAG AGT TCC TTC TTC-3' (15226)3 E3F c : (16519) 5'-TCC TGA TTT TAT TTC TGT AG-3' (16538)(55°C) E3R b : (16973 5'-ATA TCC TCC AAG GTG ACT AG-3' (169549)4 E4F c : (27765) 5'-TAG CTA GCT AAC TTC TCA AAT CTT CTA G-3' (27792)(57°C) E4R b : (28098) 5'-ATT AAC CTA GAC TGC TTC CAA GGG-3' (28075)5 E5F c : (31440) 5'-TAC AGG CTT CCA AAT CCC AG-3' (31459)(57°C) E5R b : (31707) 5'-GCT TAC CTT TAG GAT GGT GC-3' (31688)6 E6F c : (34728) 5'-CCT GCA CCT ACA AAA TCC AG-3' (34347)(57°C) E6R b : (35781) 5'-TCT GCC ATG CTA TTC AGG AC-3' (35762)7+8 E78F b : (39768) 5'-CCC TGT AGT CTC TCT GTA TG-3' (39887)(50°C) E78R c : (40200) 5'-TTA TGA GGT GCT GGA AGG AG-3' (40181)9 E9F c : (41353) 5'-AAC CCT GAC AAC TAA TAG TG-3' (41372)(57°C) E9R b : (41551) 5'-AGG ACT CCA GAT GTT TCC AA-3' (41532)a Annealing temperature within parentheses.bAfter primer name indicates that the primer is biotinylated.c After primer name indicates that the primer is Cy-5 labeled.clones, which may be comparable to the unsuccessfully expanded clones. Themutation itself, such as nonsense, frameshift, deletion, and splice mutation,may also result in low copy number of mRNA (12).Make at least two pellets for both RT- and MP-PCR, and save the remainingof the clone as “rest pellet.” Store all pellets at –80°C. Use pellets (preferablyfresh) from nonselected (wild-type) clones or previously successfully amplifiedlysates as positive controls. The possibility of using another gene (expressed ata comparable level) as an internal control for cDNA synthesis/PCR may alsobe considered. For RT-PCR-negative mutants, the “rest pellets” can besubjected to isolation of RNAs using commercially available kits. Whennecessary, the individual exons may be amplified and screened for mutation bySSCP (single-strand conformation polymorphism) before sequencing of themutated exon.