Abstract:

A novel microfluidic digital polymerase chain reaction(PCR) chip for single molecule amplification and absolute quantification of nucleic acid was fabricated by multilayer soft lithography technique and composed of three layers with valves controlling liquid, the material of silicone elastomer polydimethylsiloxane(PDMS) and glass coverslip. The microfluidic chip is equal to a piece of glass coverslip in size, which contains 4 separate panels, and each panel contains 640 independent 6 nL-chambers; the chip is capable of detecting 4 samples simultaneously. Digital PCR on the microfluidic chip was tested quantitatively using 18sRNA cDNA from A549. The sample was serially diluted and target DNA molecules were randomly distributed in chip, which can be described by Poisson distribution. If a panel contains template DNA much less than on average 0.5 template molecules per chamber, then there would be 0 or 1 copy in a chamber; the chambers containing template DNA are amplified by PCR and analyzed to be positive, while the chambers without template molecule are analyzed to be negative, the copy number of target DNA molecules of the sample can be read out accurately just by counting positive reactions. The result has proved the feasibility and flexibility of the microfluidic chip that single molecule amplification and absolute quantification of nucleic acid amplification can be succeeded. The design of the chip has the potential to meet the requirements for the general labs: inexpensive, sensitive, economizing labor time and reagent, and simple operation. It is possible to make the digital PCR technology into ordinary laboratory, and make it become one of the common tools in biology research, especially in the developing world. This technique is useful for molecular genetic analysis in cancer and infectious diseases, single cell analysis, bacterial determination, non-invasive prenatal diagnosis in which many biologists are interested.

Key words: Digital polymerase chain reaction(PCR), Microfluidic chip, Single molecular amplification, Nucleic acid quantification, Absolute quantification