Solid-Phase PCR in Microwells: Effects of Linker Length and Composition on Tethering, Hybridization, and Extension

Carmon, Amber; Mitchell, Sharon E.; Thannhauser, Theodore W.; Müller, Ulrich F.; Kresovich, Stephen

doi:10.2144/02322rr03

Cited by 24 publications

(24 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…During SP-PCR DNA, amplification occurs mostly in solution, but the increased concentration of pre-amplified target facilitates PCR on the solid support. This combination of PCR and microarray technologies were suggested as a potentially useful technique for SNP identification (12,13,15,18), allele detection (11,18), detecting infectious organisms or drug-resistant mutations (10,17,19), in vitro transcription/translation studies (21) and the quantification of SP-PCR products (14). Regardless of amplification efficiency, SP-PCR suffers from the common problem of any multiplex PCR, namely primer interference and dimer formation.…”

Section: Discussionmentioning

confidence: 99%

DNA analysis with multiplex microarray-enhanced PCR

Pemov

2005

Nucleic Acids Research

View full text Add to dashboard Cite

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3′ end of a universal amplification primer. The complex primer pair is covalently tethered through its 5′ end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 106-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 103 genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.

show abstract

Section: Discussionmentioning

confidence: 99%

DNA analysis with multiplex microarray-enhanced PCR

Pemov

2005

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…For instance, the enzymatic processing of DNA in situ has been utilized for cloning and sequencing on solid supports, enabling unprecedented high-throughput capability (Metzker 2010b). These applications require a reaction environment that provides maximal reaction efficiency, allowing nucleases to process their substrates in bulk while minimizing inhibition or side reactions imposed by crowding and steric hindrance (Carmon et al 2002;Buxboim et al 2008;McCalla et al 2009). Castronovo and coworkers studied the action of the restriction enzyme DpnII (recognition sequence, 5 0 -^GATC-3 0 ), on surface-bound dsDNA nanopatches created by nanografting .…”

Section: Enzymatic Reactions Within Nanoconfined Nucleic Acid Architementioning

confidence: 99%

Emergent Properties and Functions of Nanoconfined Nucleic Acid Architectures

et al. 2015

View full text Add to dashboard Cite

The facile ability of DNA to self-assemble has enabled the creation of complex architectures with diverse functions on surfaces or in solution. This approach provides a powerful design tool for the development of nanoscale devices with transformative applications in multiple areas, including the detection of complex biomolecules, drug delivery, and in situ biomolecular synthesis. However, little is known of the effect of confinement on the function of complex nucleic acid architectures, which exhibit unanticipated behaviors that presumably reflect high-level molecular crowding. In this chapter, we review selected recent studies that describe the application and atypical behaviors of nanoconfined nucleic acids, in particular with respect to hybridization, denaturation, conformation, stability, and enzyme accessibility. We argue that the novel behavior of dense nucleic acid arrays naturally emerge as a result of immobilization and reduction in spatial degrees of freedom. We summarize by emphasizing the need for basic physical–chemical studies of dense nucleic acid architectures, involving an interplay of experimental and theoretical approaches, in order to effectively guide the successful technological development of nucleic acid nanodevices

show abstract

Section: Enzymatic Reactions Within Nanoconfined Nucleic Acid Architementioning

confidence: 99%