e119 Nucleic Acids Research, 2010, Vol. 38, No. 11 DISCUSSION This study demonstrates that Dilute-‘N’-Go sequencing with added blockers or LPs is a convenient and accurate method for rapidly preparing and sequencing all of the oligonucleotide strands generated in a LATE-PCR multiplex reaction. In contrast, all other protocols for sample preparation prior to dideoxy sequencing are time consuming and expensive (6). The concept of preventing primer extension by addition of a competitive oligonucleotide has been used before. For instance, Gillespie turned symmetric PCR into asymmetric by stopping the reaction after 10–50 thermal cycles and adding a blocker to one of the primers (18). Nordstro¨ m et al. (19) carried out pyrosequencing from a double- stranded DNA template by adding two non-extendable oligonucleotides to prevent hybridization of both the forward and backward primers and also added an inner sequencing primer. In addition, allele speciﬁc PCR assays have been described in which a primer that is perfectly matched to the mutant but mismatch to the wild type is enhanced by addition of a blocker that is perfectly matched to the wild type strand but mismatched to the mutant strand, thereby making ampliﬁcation of wild type less eﬃcient than that of the mutant (20,21). In contrast to these previous reports the study reported here investigated the design criteria of blockers needed to achieve eﬃcient dideoxy sequencing of all strands in a multiplex LATE-PCR assay. Our results clearly show that eﬃcient blocking of each XP depends on three inde- pendent factors. First, the position of the blocker relative to the primer is important because the 30-end of the XP must be prevented from hybridization to its target to prevent elongation by the DNA polymerase. At least a single nucleotide overlap is required, but may not be suf- ﬁcient even if the BLK Tm is much higher than the XP Tm. Because hybridization is dynamic, reliable blocking requires an overlap of at least three nucleotides. Second, the Tm diﬀerence between the BLK and the XP is also critical. Blocking is eﬃcient when the Tm of the BLK is at least 5 C higher than that of the Tm of the XP. Finally, a blocker having a secondary structure with a Tm higher than the annealing temperature, typically 50 C for dideoxy sequencing, should be avoided. We are interested in detecting changes in mitochondrial genomic sequences that result from aging or drugs. But, detecting such changes is diﬃcult for two reasons. First, they are rare and second, the large number of mito- chondrial genomes in a single cell obscures sequence changes in a fraction of these genomes. The solution is to amplify and then sequence portions of individual mitochondrial genomes. But, multiplex single molecule ampliﬁcation is not all that is required. The sample must also be handled free of contamination and sequence changes that are detected in an ampliﬁed strand of DNA need to be veriﬁed since they could also be the results of errors during ampliﬁcation or during sequencing. We have recently reported that LATE-PCR is a reliable method of ampliﬁcation starting from single molecules and that Dilute-‘N’-Go of the two complementary strands of DNA generated in a monoplex reaction can be used to PAGE 6 OF 7 validate rare sequence changes (14). Diﬀerent polymerases, Platinum Taq and Tﬁ( ), have been used for LATE-PCR ampliﬁcation with no diﬀerence in the sequencing results. The HV1 and HV2 genes of the human mitochondrial genome are part of the D-loop and are known to be variable from person to person, while the Cyt b gene is conserved within a species. Preliminary data indicate that treatment of human liver carcinoma cells with AZT results in accumulation of mutations in HV2 (Osborne and Wangh, unpublished results). 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