A Method For Parallel, Automated, Thermal Cycling of Submicroliter Samples

Abstract

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 µL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer

“…Besides, 1000-fold parallelism is another great problem in handling such a minute volume of solution even if robotics could be employed. This has been a common challenge for MEMS (mechano-electro micro system) and μTAS (micro-Total Analysis System) approaches [13-15,27,28], but yet not completely solved. Therefore, a direct transfer, aperture-to-aperture (Fig.…”

“…Therefore, technologies dealing with less than μL aliquots have been pursued. Although there are not a few studies on dealing with sub-μL solutions [13-15], they are basically microfluidics approaches and are currently limited in the degree of parallelism and the number of possible reaction steps [16]. This may be because they are adopting, in a sense, a closed system so as to prevent from evaporation-loss of a tiny volume of sample, leading to rather elaborate and complicated systems.…”

Accessing genetic variation: genotyping single nucleotide polymorphisms

“…New PCR instruments that use microcapillaries instead of microtitre plate formats have been devised, and offer increased PCR throughput and reduced reagent costs as they use extremely short amplification times and small reaction volumes 121,122 . Fully automated SNP analysis systems could then be designed based on homogeneous detection, or by streamlining the PCR and the subsequent genotyping procedure in microfluidic 'lab chip' devices that operate with submicrolitre reaction volumes.…”

Nanoliter deposition on star-shaped hydrophilic–superhydrophobic patterned surfaces

“…Top performance of a non-contact dispensing system can achieve throughput up to 96 samples in a few minutes and sub-nanoliter precision, with 4-8 tips pipetting simultaneously. [12][13][14] Other methods include dip-pen nanolithography technique and polymer pen lithography, 15,16 where nanoscale tips were used to write liquid patterns directly on a substance with different kinds of inks. Impressive results have been claimed, e.g.…”