Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends

Zhang, Chunsun; Xing, Da

Several new approaches have been developed to partially or fully circumvent these problems. The roles of spiral-channel-based continuous-flow PCR devices ( Figure 2B ) have been recently emphasized by several groups since this approach can effectively avoid the possible formation of the DNA double strands (41, 42, 56, 59, 61, 71, 72, (74) (75) (76) . But it is still hard to perform parallel PCR amplifications using such devices. An oscillatory-flow-based approach ( Figure 2C ) (23, 64, 65) not only combines the cycling flexibility of the stationary chamber PCR with quick temperature transitions associated with the continuous-flow PCR, but also provides the possibility of performing high-throughput PCR amplifications in a parallel format (64) . Microfluidic digital PCR (23, 71, 72, (89) (90) (91) (92) (93) represents another example of the power of microfluidic PCR chips. This technology can provide precise control of sample volume and high-throughput analysis of serial (23, 71, 72, (89) (90) (91) or parallel (93, 92) format. In the serial format, the PCR solutions can flow continuously through a reaction channel path as a 'droplet train'. However, such systems easily suffer from cross-contamination between samples and sample dispersion, and consequently appropriate two-phase flow systems are often required (71, 72) . In spite of this shortcoming, it is still possible to implement the totally automated, contamination-free, reusable and robust microfluidic digital systems for highthroughput PCR (71) . Recently, Quake and colleagues reported another type of microfluidic digital PCR, where digital PCR was performed in parallel microarray format (92, 93) . The array chips used are commercially available, thus allowing single usage to effectively prevent carryover contamination. Importantly, the micropumps and microvalves not only can be conveniently used to distribute PCR fluid into a number of isolated reaction chambers for high-throughput PCR, but can also effectively seal the individual reaction chambers so as to realize a cross-contamination free microfluidic digital PCR system. In addition, for these microfluidic digital PCR systems, quantification relies only on binary, positive/ negative calls for each subreaction within the partitioned analyte. This affords an absolute readout of DNA copy number with single-molecule resolution (93) , allowing for transcription factor profiling in individual hematopoietic progenitors (93) and multigene analysis of individual environmental bacteria (92) . Of course, such microfluidic digital PCR can also be used for other potential applications such as detection of base substitution mutations, chromosomal translocations, alternatively spliced products. It is also useful for allelic discrimination and detection of allelic imbalance (94).