Product differentiation during continuous-flow thermal gradient PCR.
A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.
Crews
Niel
N
Department of Mechanical Engineering, University of Utah, Salt Lake City, UT 84112, USA.
Wittwer
Carl
C
Palais
Robert
R
Gale
Bruce
B
D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis* / Q000737:chemistry; D001483:Base Sequence; D041322:Chromosomes, Human, Y / Q000235:genetics; D065729:Cytochrome P-450 CYP2C9; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D055095:Optics and Photonics; D010781:Photography; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013696:Temperature
D005456:Fluorescent Dyes; D004247:DNA; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases