PAGE 3 OF 7 20–30 linear cycles is diluted to 12 ml in distilled water containing 1 mM of the same LP used in the LATE-PCR or another inner sequencing primer. For the sequencing of the XP strand, 1 ml DNA from LATE-PCR is diluted to 12 ml with 1 mM of each BLK, unless speciﬁed otherwise in the text. In order to obtain the whole sequence or the second half of the HV2 amplicon, the HV2 XP was blocked along with the other XPs in the triplex reaction and a separate downstream sequencing primer was added at 1 mM. Tm calculation Tm is deﬁned as the temperature at which, for a given concentration of primer or BLK at the start of the reaction, half of the target is bound to that primer or BLK to form a heterodimer. The Tm of each primer or BLK can be obtained from the corresponding binding curve to its target simulated by VisualOMP V7 (DNA software, CA) as shown in Figure 1b. The simulation is performed under sequencing conditions with all target concentration set as 2.5 nM, all XP concentrations set at 25 nM and all BLK concentrations set at 500 nM unless described otherwise in the text. Figure 1. Obtaining both LP strand and XP strand of G269 and HV1 from their LATE-PCR duplex by Dilute-‘N’-Go sequencing. (a) blocker design of the G269 BLK, G269_BL and HV1 BLK, HV1_BL; (b) the binding curve of each XP and BLK on its speciﬁc target and (c) sequencing results from the G269 and HV1 duplex by Dilute-‘N’-Go sequencing methods. Nucleic Acids Research, 2010, Vol. 38, No. 11 e119 RESULTS Blockers: rationale and proof of concept All of the multiplex LATE-PCR assays described here contained 50 nM of each LP and 1 mM of each XP at the start of the reaction. At the end of the exponential phase of ampliﬁcation all LPs are depleted, but each XP is still present at 500 nM after 20–30 additional cycles of linear ampliﬁcation. If an aliquot of a multiplex reaction is simply diluted into a commercial sequencing cocktail, each XP will hybridize to its speciﬁc target and will generate a ladder of oligonucleotides. All such ladders will overlap making it impossible to discern individual sequence. Therefore, to use the Dilute-‘N’-Go protocol for sequencing each of the XP strands generated in a multiplex LATE-PCR assay it is necessary to block exten- sion of all-but-one of the XPs present in the reaction. This is accomplished by adding all possible sets of all-but-one BLKs to parallel aliquots of the reaction products. In order to be eﬀective all BLKs have to: (i) speciﬁcally hybridize to their intended LP strand; (ii) overlap the 30 end of the corresponding XP and (iii) bind to their target sequences more rapidly than the corresponding XP. Figure 1a shows the simplest way to design BLKs to meet these criteria. The BLKs for both G269 and HV1 are both oligonucleotides 5–10 bases longer on their 30-ends than the XPs with which they compete. The 30-end of both BLKs is capped with a BHQ-1 (Black Hole Quencher-1) when ordered from Biosearch Technologies although a carbon linker is equally eﬀective (data not shown). Each BLK is perfectly matched to its target and therefore has a Tm at least 5 C higher than that of its corresponding XP. In silico melting proﬁles show that the two BLKs saturate the targets before the XPs as the temperature drops (Figure 1b). The Dilute-‘N’-Go sequencing results, Figure 1c, show that the addition of the HV1 BLK resulted in a clean sequence for G269 XP strand, while addition of the G269 BLK to a second aliquot resulted in a clean sequence for HV1 XP strand. Omission of both BLKs, but addition of the LP for G269 to a third aliquot generated a clean sequence for the G269 LP strand, while addition of the LP for HV1 to a forth aliquot generated a sequence for the HV1 LP stand. The sequences of the two HV1 and the two G269 strands are complementary when aligned right-to-left/left-to-right. It is worth emphasizing that preparation of the four aliquots by dilution with distilled water took only seconds to carry out. Dissecting the elements of blocker design Position eﬀect. Both BLKs in Figure 1 out compete their respective XPs by binding to the entire target sequence plus additional bases at the 30-end. But, the data shown in Figure 2 demonstrate that several BLKs with similar melting temperatures are equally eﬀective provided that they overlap with at least one nucleotide on the 30-end of the XP. In the examples shown here the lengths of all the BLKs were adjusted to keep the Tm of each BLK 10 C higher than that of the XP, 57.8 C.