e119 Nucleic Acids Research, 2010, Vol. 38, No. 11 products with one of the LPs, all of which have been depleted. The ﬁdelity of the resulting set of sequences can be conﬁrmed by comparison to each of the corre- sponding sequences generated in its own monoplex reaction (12). But, the ﬁdelity of the set of sequences derived from the multiplex reaction cannot be conﬁrmed by sequencing each of the complementary strands in the same multiplex reaction using the standard Dilute-‘N’-Go protocol. This is because all of the XPs are still present in the reaction product and can generate a set of overlapping sequences. In this paper, we introduce the use of blockers in con- junction with Dilute-‘N’-Go sequencing in multiplex LATE-PCR assays. A blocker (BLK) is designed to compete with hybridization of the 30-end of the XP to its binding site on its target, thereby preventing primer elon- gation during each thermal cycle of the sequencing reaction. All but one of the XPs is blocked in each aliquot of the diluted multiplex reaction product. Only the XP that is not blocked in that aliquot functions as a sequencing primer. The conditions for using this strategy were optimized using a duplex LATE-PCR assay and then were applied to a triplex assay comprised of three amplicons for the human mitochondrial genome. This assay is being built and tested in order to be able to monitor mutagenesis at the single genome level caused by either aging or azidothymidine (AZT), a drug used to inhibit the HIV reverse transcriptase. MATERIALS AND METHODS Synthesis and puriﬁcation of oligonucleotides Primers: G269_LP: 50-CGAGGTCATTGAATACGCACGGCTCC-30, G269_XP: 50-GCGCTGTGCCCTTACATAGTCT-30, HV1_LP: 50-CACCAGTCTTGTAAACCGGAGATGAAAACC-30, HV1_XP: 50-CGAGGAGAGTAGCACTCTT-30, TSD_LP: 50-CCGCCCTTCTCTCTGCCCCCTGGT-30, TSD_XP: 50-CCGGGTCTCTAAGGGAGAACTCCT-30, Globin_LP: 50-GGCCATCACTAAAGGCACCGAGCACT-30, Globin_XP: 50-TGGGTTTCTGATACGCACTGACTCTCTC-30, HV2_LP: 50-AGCGGTGTGTGTGTGCTGGGTAGGAT-30, HV2_LPs2: 50-GTTATGATGTCTGTGTGGAAAGTG-30, HV2_XP: 50-ACAGGTCTATCACCCTATTAACCACTCA-30, HV2_XPs2: 50-TTCTGGCCACAGCACTTAAAC-30, Cytb_LP: 50-CAGGTTAGAATGAGGAGGTCTGCGGCT-30, Cytb_XP: 50-ACCTCCTATTCTTGCACGA-30, were synthesized and salt-free puriﬁed by Euroﬁns MWG Operon (AL, USA). Blockers: G269_BL: 50-GCGCTGTGCCCTTACATAGTCTAACAGT AC-30-BHQ1, HV1_BL: 50-CGAGGAGAGTAGTACTCTTGTGCGGG A-30-BHQ1, HV1_BL1: 50-GAGGAGAGTAGTACTCTTGTGCGGG A-30-BHQ1, HV1_BL3: 50-CTTGTGCGGGATATTGATTTCACGG-30-BHQ1, HV1_BL4: 50-TGTGCGGGATATTGATTTCACGGAG G-30-BHQ1, PAGE 2 OF 7 HV1_BL6: 50-CGAGGAGATTAGTACTCTTGTGCGGG A-30-BHQ1, HV1_BL7: 50-CGAGGAGATTAGTACTCTTATGCGGG A-30-BHQ1, HV1_BL8: 50-CGAGGAGATAAGTACTCTTATGCGGG A-30-BHQ1, HV1_EB: 50-CGAGGAGAGTAGCACTCTT-30-BHQ1, TSD_BL: 50-CCGGGTCTCTAAGGGAGAACTCCTGCTCTC A-30-BHQ1, TSD_BLm: 50-CCGGGTATCTAAGGGAGAACTCCTGCTCTC A-30-BHQ1, Globin_BL: 50-TGGGTTTCTGATAGGCACTGACTCTCTCTGCCTA TTGG-30-BHQ1, HV2_BL: 50-ACAGGTCTATCACCCTATTAACCACTCAC G-30-BHQ1, Ctyb_BL: 50-ACCTCCTATTCTTGCACGAAACGGGATC A-30-BHQ1, were synthesized and double HPLC puriﬁed by Biosearch Technologies (CA, USA). LATE-PCR conditions All PCR reactions had the following reagents: 1 PCR buﬀer, 0.2 mM dNTPs, 3 mM MgCl2, 2 units of Platinum Taq DNA polymerase (InVitrogen, CA, USA), 50 nM of each LP, 1 mM of each XP and 0.24 SYBR Green I in a 25 ml volume. For G269 & HV1 duplex and TSD & Globin duplex, 300 nM PrimeSafeTM I (12) and 1000 genomes of puriﬁed human genomic DNA were included. For mitochondrial DNA HV1, HV2 and Cyt b triplex, 300 nM PrimeSafeTM II and 100 K copies of mitochondrial DNA from puriﬁed human DNA were included. LATE-PCR ampliﬁcation and ﬂuorescence detection were carried out in a Stratagene Mx 3005 PCR thermocycler. Thermal cycling proﬁle for G269 & HV1 duplex and TSD & Globin duplex included an initial denaturation step of 5 min at 95 C, followed by 55 cycles of 95 C for 10 s, 64 C for 20 s and 72 C for 1 min, then a melting from 55 to 95 C with a 1 C increase. The thermocycler temper- ature program for the mitochondrial DNA HV1, HV2 and Cyt b triplex consisted of denaturation at 95 C for 5 min, followed by 60 cycles of 95 C for 30 s, 62 C for 20 s and 72 C for 45 s, then a melting from 35 to 95 C with a 1 C increase. The completed LATE-PCR samples were kept at 20 C until sequenced. The ﬂuorescence reading in all assays was taken at 72 C. Sample preparation for dideoxynucleotide sequencing No puriﬁcation of the LATE-PCR product was per- formed. Dideoxy sequencing was performed by GENEWIZ, Inc (South Plainﬁeld, NJ) using Applied Biosystems BigDye version 3.1. The reactions were then run on Applied Biosystems’s 3730x1 DNA analyzer. Standard cycle sequencing includes an initial denaturation at 96 C for 1 min, followed by 25 cycles of 96 C for 10 s, 50 C for 5 s and 60 C for 1 min. The premixed samples were diluted 2 in GENEWIZ before the reaction. For the sequencing of the limiting primer strand, 1 ml DNA from a 10 diluted LATE-PCR that has completed