Analysis of Specific Gene by Integration of Isothermal Amplification and Electrophoresis on Poly(methyl methacrylate) Microchips

Abstract

We have successfully achieved the integration of isothermal amplification and the subsequent analysis of specific gene fragments on poly(methyl methacrylate) microchips. In our experiments, loop-mediated isothermal amplification, which can offer higher specificity and efficiency than PCR, has been performed at a constant temperature (65°C ). After amplification, products could be either examined by the integrated microchip-based electrophoresis or directly observed by naked eye with SYBR Green I added into the reaction solution. By such an integrated microsystem, the amplification and the subsequent analysis of prostate-specific antigen gene with template concentration at 23 fg/µL could be finished within 15 min, which demonstrates its advantages of high specificity, good reproducibility, and fast speed in gene detection.Sequencing of the human genome has been almost completed, and the human genome project quickly moves on to the postgenome-sequencing era. [1][2][3][4] During this period, further development of analytical technology is highly required for high-throughput screening of disease-causing genes and high-speed analysis of genetic polymorphism on an individual genome. Accordingly, the development of novel microchip-based methods for ultrafast DNA analysis has been challenged, especially for the amplification and analysis of genetic locus on the genome. [5][6][7][8] Since its initial study from the middle of 1980s, PCR has been playing an important role in modern biology research. 9 During the amplification, thermal cycling with temperature ranging from 50 to 100°C is indispensable. Therefore, most reported PCR chips are fabricated by either glass or silicon. 10-13 However, polymer microchips, which are expected to be very promising in the future, 14 can hardly endure the high temperature during PCR. Accordingly, amplification at a relatively low temperature should be especially developed for them.Up until now, several isothermal amplification methods under mild conditions have been proposed, including nucleic acid sequence-based amplification, 15 self-sustained sequence replication, 16 and strand displacement amplification. 17,18 Each of them has its own innovation to reinitiate new rounds of DNA synthesis, but there are still drawbacks to overcome. 19,20 They require either a precision instrument for amplification or an elaborate method for detecting the amplified products due to poor specificity of target sequence selection. Loop-mediated isothermal amplification (LAMP) is another newly invented method that can amplify a few copies of DNA to 10 9 within 1 h under isothermal condi-* To whom correspondence should be addressed. Phone: +86-411-83693459.

Microfluidic Chips for Point‐of‐Care Immunodiagnostics

“…To circumvent this challenge, nucleic acid amplifi cation reactions have been developed to operate at reduced or constant temperatures. Loopmediated isothermal amplifi cation (LAMP) uses four specially designed primers and a DNA polymerase to amplify nucleic acids at a constant temperature of ∼ 60 ° C. [ 137 ] LAMP has been integrated onto microfl uidic chips for the analysis of genes [ 138 ] and detection of pathogens. [ 139 ] In self-sustained sequence replication (3SR) [ 140 ] and nucleic acid sequence based amplifi cation (NASBA), [ 141 ] a set of transcription and reverse transcription reactions amplify the nucleic acid.…”

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

“…Thus, many microsystems that combined LAMP with different detection methods, newly designed lab-on-chips, microfluidic cartridges and automated techniques were reported. In many studies, the LAMP amplification signals were already detected after 15-40 minutes after the thermal reaction started [50][51][52][53][54].…”

“…Thus, many microsystems that combined LAMP with different detection methods, newly designed lab-on-chips, microfluidic cartridges and automated techniques were reported. In many studies, the LAMP amplification signals were already detected after 15-40 minutes after the thermal reaction started [50][51][52][53][54].The preferred and the most sensitive method for detection is based on fluorescence (with SYBR green, SYTO green or EvaGreen as fluorescent dyes) that can be detected as early as 15 minutes after the thermal reaction starts. For calcein, a limit of detection (LOD) of about 270 copies/µL was described [55].…”

Nanomaterials and chip‐based nanostructures for capillary electrophoretic separations of DNA

“…Numerous capillary electrophoresis (CE)-and microchip capillary electrophoresis (MCE)-based approaches have been tested and validated for DNA sequencing [1,2], genotyping [3,4], mutation analysis [5,6], characterization of single nucleotide polymorphisms (SNPs) [7,8], forensic human identification [9,10], diagnosis of diseases [11,12], and other applications [13,14]. When compared to slab-gel electrophoresis, these techniques provide the advantages of comparable resolving power, rapidity, high throughout, minute sample requirement, and ease of integration [10,15,16]. Using these techniques, small separation channels/capillaries are often filled with polymer solutions that are advantageous over cross-linked gels, including relatively low viscosity, ease of preparation, and flexibility.…”