title: Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing.
abstract: This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.
authors.affiliation: Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Rice
authors.forename: J Aquiles
authors.initials: JA
authors.lastname: Sanchez
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Pierce
authors.forename: Arthur H
authors.initials: AH
authors.lastname: Reis
authors.forename: Adam
authors.initials: A
authors.lastname: Osborne
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D052160:Benzothiazoles; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000032:analysis; D003959:Diamines; D007202:Indicators and Reagents; D007700:Kinetics; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004277:DNA, Single-Stranded; D003959:Diamines; D007202:Indicators and Reagents; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA