e119 Nucleic Acids Research, 2010, Vol. 38, No. 11 PAGE 4 OF 7 Figure 2. Eﬃciency of blockers in correlation with the position where the blocker binds to the target. Here the blockers from top to bottom are HV1_BL, HV1_BL1, HV1_BL3 and HV1_BL4, which have 19, 18, 3 or 1 nucleotide overlap with the 30-end of HV1 XP, respectively. Figure 3. Eﬃciency of blockers in correlation with the Tm of the blocker binding on its target. Here the blockers from top to bottom are HV1_BL, HV1_BL6, HV1_BL7 and HV1_BL8, which have zero, one, two or three mismatches with the target, respectively. This guarantees that as the temperature is lower the BLK is positioned on the target before the XP has a chance to bind. Figure 2 shows the G269 XP strand sequence in the absence or presence of one of several possible HV1 BLKs. When no BLK was added, both the G269 and HV1 sequences are generated and overlap. When only a single nucleotide of the HV1 BLK overlaps the 30-end of XP the base call of the G269 sequence is accurate, although there is a slight background of the HV1 sequence. Additional experiments, however, demonstrate that blocking does not occur if the blocker binds to the target beyond the 30-end of the XP (data not shown). Blocker Tm eﬀect. In order to examine the relationship between BLK Tm and XP Tm independently of other factors, we designed four BLKs of the HV1 XP that have zero, one, two or three mismatches with the corre- sponding target, Figure 3. Each of the mismatches causes the Tm of the BLK to decrease a few degrees. One HV1 BLK, HV1_BL, is perfectly complementary to the target and has a Tm 14 C higher than that of the HV1 XP. The target is completely bound by the BLK prior to binding the XP. The Tm of the BLK with one mismatch, HV1_BL6, is 66 C. While BLKs with two mismatches, HV1-BL7, and with three mismatches, HV1_BL8, have Tms of 60 and 57.2 C respectively, which are just 2 C higher or even lower than the Tm of HV1 XP. As shown in Figure 3, a clean G269 sequence was obtained when the Tm of the BLK was more than 8 C above the Tm of HV1 XP. But, the G269 sequence was not free of interfering sequences when the Tm of the BLK was only 2 C higher than the Tm of the HV1 XP. Concentration eﬀect. The melting temperature of two complementary oligonucleotide strands depends in part on their concentrations (15–17). Tm of an XP is higher at high concentration than it is at low concentration. Each XP in the HV1 & G269 duplex reaction is present at 1000 nM and is estimated at 500 nM after 20–30 cycles of linear ampliﬁcation. When the ampliﬁcation products are then diluted and sequenced using the Dilute-‘N’-Go protocol the ﬁnal concentration of the XP is 25 nM. Overall this translates into a decrease in the Tm of the HV1 XP from 64 to 57.8 C. We designed an HV1 BLK, HV1-EB, which has the same sequence as the XP but capped at the 30-end (Figure 4a). The Tm of HV1-EB increases from 53.5 to 65.9 C when its concentration is raised from 2.5 nM to 5 mM. Figure 4b demonstrates that the G269 sequence from the G269 XP becomes cleaner as the concentration of HV1-EB increases. At a concentration of 500 nM the BLK Tm is 4.6 C higher than the XP Tm and the G269 sequence is clean. These results are consistent with those of Figure 3 and allow us to conclude that a Tm diﬀerence of 5 C between the BLK and the XP is suﬃcient for full blocking, except as described below. Secondary structure eﬀect. In yet another LATE-PCR assay we used the TSD & Globin amplicons (see ‘Materials and Methods’ section) to test the eﬃcacy of blockers in a duplex reaction, Figure 5. The Tm of TSD XP is 67 C while the Tm of Globin XP is still 61 C. BLKs, TSD-BL and Globin-BL, were designed with Tms about 10 C higher than their corresponding XPs. The sequencing results for the two XP strands in this duplex are shown in Figure 5a. As expected both sequences are present in the absence of any BLK. In contrast, TSD XP generated a clean sequence in the presence of the Globin-BL. But, Globin XP did not generate a clean sequence in the presence of the TSD-BL. Further analysis revealed that the TSD-BL forms a hairpin at 50 C, Figure 5b, which renders it ineﬀective as a blocker. We therefore modiﬁed the blocker sequence by introducing a mismatch which destabilized the stem. Blocker TSD_BLm, Figure 5c, does not form a hairpin at 50 C and eﬃciently blocks the TSD excess primer as shown in Figure 5a.