| IntroductionCurrently,great attention has been focused on the molecular diagnosis,therapy and prophylaxis of gene-based diseases such as cancers.Single-nucleotide polymorphisms(SNPs)/mutations and methylation,being the most common form of genetic and epigenetic variation,often offer markers of the highest resolution for tracking disease genes.DNA sequencing is considered the gold standard for DNA analysis,while the high-cost and laborious operations prevent it from being employed universally.Nowadays,although many techniques have been developed for detecting mutations and methylation,increasing efforts are being aimed at developing techniques that offer greater speed,accuracy,and cost-effectiveness than what is currently available.Temperature-gradient gel electrophoresis(TGGE)offered an effective approach for detecting SNPs and mutations without resorting to more sophisticated DNA sequencing procedures.In TGCE,where electrophoresis is processed under a continuous temperature gradient,the DNA fragments consisting of homoduplexes and heteroduplexes are separated according to the difference of their melting temperatures. Compared to other presequencing screening methods,the most outstanding advantage of TGCE is that it can detect mutations without knowing their exact nature,as long as the Tm's of the samples studied are sufficiently close to the applied temperature gradient range.However the traditional gel electrophoresis and the complexity of temperature gradient system causes poor reproducibility and low detection sensitivity. Microfluidic analysis system is a micro-biological and chemical analysis tools based on physics,analytical chemistry and biology.An integrated microfluidic device incorporates many of the necessary components and functionality of the typical room-sized laboratory on a small chip,also called"lab-on-chip".Microfluidic chip offers several advantages over traditional gel electrophoresis,the most important of which are high-throughput capabilities and high sensitivity,reduced sample and solvent consumption,increased portability,and reduction in analysis time.More recently,TGGE has been implemented in capillary electrophoresis(TGCE) and microchip formats(chip-based TGCE)as an alternative to slab-gel electrophoresis. The advantages of higher separation efficiency with chip-based systems were achieved mainly owing to smaller injected sample lengths and higher dissipation of Joule heating, allowing application of higher field strengths compared with CE.In this work,we developed a simple,cheap and robust system for establishing stable and reproducible spatial temperature gradients on a simple glass microfluidic chip for TGCE separations,without resorting to complicated equipment and fabrication techniques,and which is easily adaptable to standard microfluidic CE chips.The chip-based TGCE system can successfully detect the K-ras mutation and p16 methylation,providing a more effective analytical tool for tumor-associated genes screening.Materials and Methods1,Establishment of chip-based TGCE system for DNA mutation analysis(l)Microchips were fabricated by using standard photolithography and wet chemical etching techniques.The etched plate and the cover plate were bonded together via fusion bonding.A home-build LIF detection device and a PMT detector were employed for detection.(2)A home-made thermostated heater plate was used to provide gradient heating along the separation channel of the glass chip.The heater plate was mounted on the chip,and a continuous thermal gradient could be formed by slanting the heater relative to the plane of the glass chip by inserting a thin spacer at one end of the plate.During the electrophoresis analysis,the DNA sample was electrokinetically introduced into the sample channel and separation channel.(3)Four known point mutation samples were detected under the temperature gradient of 5℃(1.7℃cm-1)and 10℃(3.3℃cm-1)in less than 7min.In this study,a much shorter heating length of only 3.0 cm(4.5 cm separation length)was employed, and found to be sufficient for resolving all samples studied.2,K-ras mutation detection with chip-based TGCE(l)The point mutation of K-ras gene from 6 colon cancer cell lines was tested by chip-based TGCE.(2)According to coden 12 of K-ras gene,a mixed model of wild and mutation with proportions of 1:1,4:1,16:1,64:1,128:1,256:1,512:1 was established and was detected by chip-based TGCE,observing the detection limit for mutant K-ras.(3)K-ras gene of 84 Paraffin-embedded tissues from patients with colorectal cancer was detected by chip-based TGCE system.Compared with sequencing,the relationship between K-ras mutation rate and colorectal cancer screening was analysized.(4)K-ras gene of 20 tumor tissues and paired fecal samples from patients with colorectal cancer were detected by chip-based TGCE system.Compared with sequencing,detection rate and accordance rate for K-ras mutation were analysized.(5)AutoCAD software was applied to get the peak area of homoduplexes and heteroduplex.To determine the ratio of mutant K-ras,the peak height for mutant K-ras was divided by the sum of the peak heights for wild and mutant.The non-adjusted proportion of mutant K-ras from chip-based TGCE and the ratio of the mutant clones to the total number of sequenced clones were used to calculate an exact proportion of mutant K-ras. 3,p16 methylation detection with chip-based TGCE(l)Genomic DNA is treated with bisulfite prior to PCR amplification using primers encompassing differentially methylated sequence,whereby multi-mutations are introduced by conversion of cytosines to thymines in the unmethylated CpG sites and no conversion in the methylated ones,finally all methylation patterns can be identified by electrophoretic peak profile.(2)Fragment of p16 with various numbers of methylated sited were mixed with unmethylated fragment,and then were detected by chip-based TGCE under the temperature gradient of 5℃and 10℃in a shorter heating length of only 3.0 cm。Complex methylated samples were detected under these temperature gradients.(3)The p16 promoter methylation from 5 known colon cancer cell lines and 4 unknown colon cancer cell lines were tested by chip-based TGCE.(4)According to promoter methylation of p16,PCR products from unmethylated and methylated colon cell lines were mixed at ratio of 1:1 and were diluted 50 times, 100 times,200 times,400 times,800 times to detect the detection limit for DNA molecules.A mixed model of unmethylated and methylated PCR products with proportions of 1:1,4:1,16:1,64:1,128:1,256:1,512:1 was established and was detected by chip-based TGCE,observing the detection limit for methylated fragment.(5)To further study the applicability of this chip-based TGCE system to clinical samples,p16 promoter methylation of 20 tumor tissues and paired fecal samples from patients with colorectal cancer were detected by chip-based TGCE system.Results1,The slantwise radiative heating system could provide stable and uniform temperature gradients required for performing mutation detection on chip-based TGCE with simple instrumentation.Four known point mutation samples were successively detected under the temperature gradient of 5℃and 10℃in a shorter heating length of only 3.0 cm.The recordings showed good reproducibility in peak migration time(1.9% RSD,n=5)and peak pattern. 2,The point mutation of K-ras gene from 6 colon cancer cell lines and all kinds of clinical samples were successively tested by the present system.The detection limit for mutant K-ras is 1/513.3,Various mathylation model samples were successively detected with this chip-based system.The recordings showed good reproducibility in peak migration time (RSD 1.7%under 5℃temperature gradient,RSD 0.9%under 10℃temperature gradient,n=5)and peak pattern.The detection limit for DNA molecules is 0.5ng/μl, and the detection limit for methylated pl6 is 1/257.The methylation state of pl6 gene from 7 colon cancer cell lines and all kinds of clinical samples were successively tested by chip-based TGCE.ConclusionsIn this study,we showed the effectiveness of a robust and simply constructed slantwise radiative heating system for chip-based TGCE.Efficient and reproducible detection of genetic point mutations and methylation in DNA samples was achieved on the basis of stable and uniform spatial temperature gradients established along the separation channel.The system allows effective mutation and methylation detection within a wide temperature range of 10℃within a short separation length of only 3 cm. This is important for broadening the range of applicability for detection unknown mutations and methylations.Therefore,the approach shows promise in developing dedicated microfluidic equipment for early diagnosis of cancer diseases involving known gene mutations and methylation.
|
PDF Full Text Request