Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing.
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.
Rice
John E
JE
Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Sanchez
J Aquiles
JA
Pierce
Kenneth E
KE
Reis
Arthur H
AH
Osborne
Adam
A
Wangh
Lawrence J
LJ
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
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