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

Zhang, Chunsun; Xing, Da

All existing DNA detection methods can be used for off-line detection of on-chip PCR products. The use of intercalators in combination with gel electrophoresis is the most widely used method for the detection of post-PCR products (14, 23, 25, 28, 30, 38, 40, (44) (45) (46) (47) (48) 50, 51, 54, (56) (57) (58) (59) (60) (62) (63) (64) 66, 70, 72, 77, 89) . In this combination, the DNA molecules are effectively labeled with an intercalating dye and subsequently separated according to their sizes. Upon binding to double-stranded DNA, the intercalator molecules exhibit significant enhancement in their fluorescence quantum efficiencies. Ethidium bromide (EtBr) and SYBR Green I are the most popular intercalator dyes, but other intercalating dyes, such as GoldView TM , have also been investigated (74) (75) (76) . The use of intercalator to detect DNA molecules has two main advantages: real-time detection (10, 20, 77, 79, 80) and versatility. However, indiscriminate binding is also a major disadvantage: both specific and nonspecific PCR products can produce the same type of signal and it is difficult to differentiate between them. In addition, this technique is time-consuming and labor intensive. CE is another widely used off-line detection technique (97) (98) (99) (100) . The use of CE for DNA separation detection has several obvious advantages including low operating costs, high separation efficiency, small sample volume, short analysis time, versatility and simplicity. Incorporation of CE on a chip fully utilizes these advantages. CE chips were first demonstrated in 1990 by Manz et al. (101) and have now become commercially available. One notable example is the Agilent 2100 Bioanalyzer (Agilent Technologies). Recently, this commercial CE chip has been used for off-line detection of PCR products (10, 11, 13, 16, 69, 73) . Other microchip electrophoresis systems include the Hitachi SV 12-channel electrophoresis microchip (Hitachi Electronics Co.) (73) , LabChip Õ 90 Automated Electrophoresis System (Caliper Lifesciences) (20) and CE chips with laserinduced fluorescence (LIF) detection (41, 52, 53, 56) . The off-line CE chip platform can offer walkaway and unattended analysis of DNA molecules, eliminate the time-consuming and messy slab gel process, generate much more reproducible and high quality data, and allow high-throughput laboratory analysis; however, the manual sample loading may increase the risk of cross contamination and the total analysis time.