Published online 26 February 2010 Nucleic Acids Research, 2010, Vol. 38, No. 11 e119 doi:10.1093/nar/gkq111 Dilute-‘N’-Go dideoxy sequencing of all DNA strands generated in multiplex LATE-PCR assays Yanwei Jia, Adam Osborne, John E. Rice and Lawrence J. Wangh* Biology Department, Brandeis University, 415 South St., Waltham, MA 02155, USA Received August 20, 2009; Revised February 2, 2010; Accepted February 8, 2010 ABSTRACT We have recently described a Dilute-‘N’-Go protocol that greatly simplifies preparation and sequencing of both strands of an amplicon generated using linear-after-the-exponential (LATE)-PCR, an advanced form of asymmetric PCR . The same protocol can also be used to sequence all limiting primer strands in a multiplex LATE-PCR, by adding back each of the depleted limiting primers to a separate aliquot of the multiplex reaction. But, Dilute-‘N’-Go sequencing cannot be used directly to sequence each of the excess primer strands in the same multiplex reaction, because all of the excess primers are still present at high concentra- tion. This report demonstrates for the first time that it is possible to sequence each of the excess primer strands using a modified Dilute-‘N’-Go protocol in which blockers are added to prevent all but one of the excess primers serving as the sequencing primer in separate aliquots. The optimal melting temperatures, positions and concentrations of blockers relative to their corresponding excess primers are defined in detail. We are using these technologies to measure DNA sequence changes in mitochondrial genomes that accompany aging and exposure to certain drugs. INTRODUCTION A host of novel sequencing methods are being developed for rapidly sequencing whole genomes by in silico splicing of short overlapping DNA sequences (1–5). Nevertheless, dideoxy sequencing remains the method of choice for sequencing PCR amplicons up to 1000 bases long, even though preparation and clean-up of such amplicons prior to sequencing is still a time consuming and expensive bot- tleneck to high-throughput dideoxy sequencing. The con- ventional approach to sample preparation is to remove unused primers, nucleotides and enzyme present at the end of ampliﬁcation by ﬁltration, chromatography or gel electrophoresis and then add a standard sequencing cocktail (6). Enzymatic methods have also been employed for the removal of interfering components (7–9). None of these procedures, however, solves the problem of sequencing all of the strands generated in a single tube multiplex reaction. Linear-after-the-exponential (LATE)-PCR is an advanced form of asymmetric PCR that uses a limiting primer (LP) and an excess primer (XP) to exponentially amplify double-stranded DNA eﬃciently and then pre- dictably switches to linear ampliﬁcation of single-stranded DNA (10,11). Limited amount of LP strand, the DNA strand extended from the LP, has been generated. While the LP is no longer present at the end of the reaction and a high concentration of the XP strand has accumulated, unused XP is still abundant. The LP strand can readily be sequenced by simply diluting an aliquot of the reaction into distilled water containing a few picomoles of the LP used for ampliﬁcation of the double-stranded amplicon. The solution is so diluted that the LP strand as the template for sequencing the XP strand has been diluted out. Therefore, no XP strand sequence can be obtained. The added LP primer serves as the sequencing primer while the XP strand serves as template when an aliquot of the mixture is added to a cocktail for commercial dideoxy sequencing (12). We have also adapted this approach for pyrosequencing (13). In the case of dideoxy sequencing, a second aliquot of a monoplex reaction can be diluted and added to a commercial sequencing cocktail without addition of the LP. In this case, the XP still present in the reaction serves as the sequencing primer and the LP strand serves as template. We have recently shown that sequencing carried out in this way is accurate, even when PCR ampliﬁcation is initiated with as little as a single DNA molecule (14). Multiplex LATE-PCR assays generate separate single-stranded XP strand for each of the amplicons. Each of these strands can be used as a sequencing template by spiking separate aliquots of the reaction *To whom correspondence should be addressed. Tel: +1 781 736 3110; Fax: +1 781 736 3107; Email: wangh@brandeis.edu The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.