PAGE 5 OF 7 Nucleic Acids Research, 2010, Vol. 38, No. 11 e119 Figure 4. Eﬃciency of blockers in correlation with the concentration of the blocker. (a) HV1 blocker, HV1_EB, has the same sequence of XP with a BHQ-1 at the 30-end and (b) G269 XP strand sequence obtained from the G269 and HV1 duplex with diﬀerent concentrations of the HV1 BLK included in the Dilute-‘N’-Go sequencing. Figure 5. Sequencing from duplex of TSD and Globin. (a) sequencing from the duplex with Globin or TSD BLK; (b) the secondary structure of TSD-BL and (c) the secondary structure of TSD-BLm, a modiﬁed TSD blocker with a nucleotide mismatch to its target. Demonstration of the utility of blockers in a triplex LATE-PCR assay We are interested in detecting mutations that accumulate in mitochondrial genomes due to aging, diabetes and envi- ronmental damage. Because these mutations are rare it is important to sequence both strands of each amplicon to Figure 6. Sequence of all six strands of mitochondrial DNA HV1, HV2 and Cyt b by Dilute-‘N’-Go sequencing. The blockers used here were HV1_BL, HV2_BL and Cytb_BL. HV2_LPs2 and HV2_XPs2 were used to get the second part of the HV2 LP or XP strand. verify that changes in one strand are not due to sequencing errors (14). In order to pursue this inquiry we have built a triplex LATE-PCR assay for three genes in the human mitochondrial genome HV1, HV2 and Cyt b (see ‘Materials and methods’ section). The amplicons generated in this reaction are 425–541 base pairs long and Figure 6 shows the results of sequencing all six strands using the Dilute-‘N’-Go protocol with appropriate BLKs. Three aliquots of the reaction were used to sequence the LP strands by adding back the correspond- ing LPs without any BLK. Three additional aliquots were used to sequence the XP strands by adding two BLKs that prevented extension of the remaining two XPs in the reaction. But, sequencing the whole HV2 amplicon failed because it contains a string of Gs (or Cs) followed by a string of Ts (or As). Only the sequence before the Gs (or Cs) were obtained. Extensive trials with Genewiz, Inc established that this was a sequencing problem indepen- dent of the Dilute-‘N’-Go protocol. To circumvent this problem we designed an inner HV2 sequencing primer, HV2_LPs2, which hybridizes to the HV2 XP strand down- stream of the string of Cs. It was added to an aliquot of the reaction instead of the LP used for LATE-PCR ampli- ﬁcation and it generated the downstream portion of the relevant sequence. To sequence the second part of HV2 XP strand we blocked all three of the XPs used for ampli- ﬁcation and added an inner sequencing primer, HV2_XPs2, which hybridizes downstream of the string of Ts in its template. The sequence of the whole HV2 amplicon can be obtained by combining the ﬁrst part driven by HV2 LP (or XP) and the second part driven by HV2 LPs2 (or XPs2). Clean sequences for all strands in the triplex LATE-PCR reaction are obtained as shown in Figure 6.