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* E-mail: yzfang@chem.ecnu.edu.cn Received September 27, 2009; revised and accepted February 23, 2010.

Project supported by Science and Technology Commission of Shanghai (No. 08142201400) and New Century Excellent Talents in University.

Chin. J. Chem. 2010, 28, 797—802 © 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 797

Multiplexed p53 Mutation Detection by Microchip Electro- phoresis with Laser-Induced Fluorescence Detector

Li, Ganga(李钢) Ge, Shulia(葛淑丽) Ni, Xiaofanga,b(倪晓芳) Wang, Qingjiang*,a(王清江) He, Pinganga(何品刚) Fang, Yuzhi*,a(方禹之)

a Department of Chemistry, East China Normal University, Shanghai 200062, China b Department of Chemistry, Ecole Normale Super Paris, Paris, 75001, France

A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7, 8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electro-phoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the applica-tion of SSCP and ME analysis as a rapid and effective method to the detection of mutations for exons 7, 8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 4 mg/mL poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7, 8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7—9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n＝5). The detection limit can be as low as 1 ng•L－1.

Keywords microchip electrophoresis, p53 gene, polyethyleneoxide

Introduction

Single-nucleotide polymorphisms (SNPs) are genetic changes resulting from single-nucleotide substitutions. For example, mutations in the p53 gene have been im-plicated in a wide variety of human cancers,1 with mis-sense mutations comprising a large majority of delete-rious p53 sequence alterations. Furthermore, point mu-tations are suspected to be responsible for complex dis-eases such as heart disease and psychiatric disorders. So detection of the point mutations has become a central issue.

A wide variety of techniques have been used to de-termine the exact location and nature of mutations in p53 gene, and many of these methods have recently been reviewed.2,3 One such approach involves isotopic 15N or 13C labeling of specific nucleobases within an oligonucleotide and determining the amount of damage at the site by mass spectrometry.4,5 Although this method is rapid and also identifies the type of nucleo-base adduct formed at the specific site, isotope-labeled oligonucleotides are up to 100-fold more expensive than fluorescent-labeled oligonucleotides. Also, with isotope labeling, it is only practical to characterize previously identified specific mutation sites, and the method is lim-ited by the size of the labeled oligonucleotides.

Another method is temperature gradient capillary electrophoresis (TGCE),6 which reveals a mutation

based on the difference in melting temperature between homoduplex and heteroduplex DNAs. Although close to 100% of point mutations can be detected in slab-gel applications, this mode of detection is labor intensive and time-consuming.

A form of single-strand DNA-conformation poly-morphism analysis (SSCP) is widely used to detect and analyze mutations and polymorphisms.7,8 Aside from its quantitative nature, one prominent advantage of SSCP over other methods is the simplicity of the sample preparation protocol. Oligonucleotide samples are sim-ply diluted and then subjected to electrophoresis. SSCP employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis.9 To bring this tech-nique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation.10,11 However, the resolving power of SSCP, a prerequisite basis for reliability re-quired in diagnostics, remains as a challenge for CE systems.

Current trends in development of new-style analyti-cal system have shifted toward the creation of miniatur-ized total analysis system for the following advantages compared to the conventional CE: reduced sample and reagent consumption, shorter analysis time, higher sen-sitivity, and portability. Recently, the development of microchip electrophoresis (ME)12,13 has played an im-portant role in the modern applications of DNA analy-

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sis.14 It is a technique that can integrate multiple sam-ples to handle processes with an actual measurement step, to increase analysis speed, negligible sample con-sumption, and to enhance miniaturization, high sample throughput.15,16 Recently, using a series of functional-ized beads in a single microfluidic channel, low-abun- dant point mutations in p53 gene were detected.17 However, functionalization of microbeads needed time consuming operations and the microchannel could be blocked by the microbeads.

Owing to its inherent selectivity and excellent sensi-tivity, laser-induced fluorescence (LIF) has been be-coming the most popular detection scheme in conjunc-tion with ME. Tian et al.18 first demonstrated mutations in BRCA1 and BRCA2 genes were examined on a mi-crochip with LIF detector. They were able to detect three different mutations in less than 2.5 min on the mi-crochip. The aim of our study was to further explore the possibility of using SSCP-ME with LIF detector for detection of mutations at exons 7, 8 and 9 of p53 gene. Advantages of this method included low cost, simple operation, and fast separation. Under the optimized conditions, the mutant and normal ssDNA were sepa-rated within 60 s.

Experimental

Equipment

The used ME system coupled with LIF detection de-vice was made by Shanghai Spectrum Company, Zheji-ang University and our research group together.19 A diode laser was used to generate an excitation beam at 488 nm and an incident power of about 4 mW. The laser beam was focused by a concave mirror, illuminating at 45° from below the chip, on the detection point of the chip channel, with a focus point of about 70 µm. The fluorescence was collected by the objective, filtered by a bandpass filter to the photomultiplier tube (PMT). A white beam from the illuminator on the microscope il-luminated the microchip. It was then focused by the objective, reflected by a slide in-and-out mirror. The high-voltage power unit variable in the range 0—6000 V was used for on-chip sample injection and gel elec-trophoretic separations. The amplified currents were transferred directly through a 10 kΩ resistor to a 24-bit A/D interface at 10 Hz (Borwin, JMBS Developments, Le Fontanil, France) and stored in a personal computer.

Fabrication of glass chips

Photolithographic and wet chemical etching tech-niques were used for fabricating microchannels onto a 1.6 mm-thick 20 mm×60 mm glass substrate. Type SG2506 glass substrate with chromium and AZ1805 photoresist coating and glass cover plates of the same dimensions were obtained from Shaoguang Microele-tronics Corp. (Changsha, China). Production of the photomask and procedures for fabrication of micro-channels were previously described. Briefly, a design on

a photomask with microchannels was transferred onto the glass plate following a UV exposure. The micro-channels were etched into the plate in a well-stirred bath containing dilute HF/NH4F. Four 1.2 mm-diameter ac-cess holes were drilled on the etched plate at channel terminals using an emery drill-bit, forming four reser-voirs. Microchips with a crossed-channel design were used for their performance in MCE separations. As shown in Figure 1A, the channel dimensions were 20 µm deep and 70 µm wide. The separation channel (from reservoir 1 to reservoir 3) was 60-mm long and was a 45-mm effective separation channel. The bonding proc-ess of the glass microchips was previously reported.20

In our experiment, the glass substrate and cover plate were just rinsed using deionized water, then the surfaces of glass substrates and cover plates were closed posi-tioned together directly for bonding at room temperature. In contrast to previous work,20 the labor involved was minimum and the procedure was much safer.

Figure 1 (A) Layout of the microfluidic chip used for the sepa-ration of double-strand DNA. Reservoir 1＝buffer inlet, reservoir 2＝sample outlet, reservoir 3＝buffer waste, reservoir 4＝sample inlet. (B) Schematic diagram illustrating the general principles of the ME-SSCP.

Chemicals

All reagents were commercially available and used without further purification. Water was purified using Milli-Q instrument (Millipore, MA, USA). The DNA concentration was determined by measuring the ab-sorbance at 260 nm. A mixture of equal amounts of chemically synthesized 20 mer ssDNA (normal ssDNA) and its single-base-substituted 20 mer ssDNA (mutant ssDNA) were used as sample. The sample was labelled in 5' with Carboxyfluorescein (FAM)21 and was diluted to 1 ng•µL－1 with a 1×TBE buffer prior to use. The mutant and normal ssDNA in this study were purchased from Sangon Biological Engineering Technology Ser-

Multiplexed p53 Mutation Detection by Microchip Electrophoresis

Chin. J. Chem. 2010, 28, 797—802 © 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 799

vices (Shanghai, China) with the following sequences: 5'-FAM-GTGATGCTGGTGAGGATGGG (in the p53 gene of normal exon 7) 5'-FAM-GTGATCCTGGTGAGGATGGG (in the p53 gene of mutant exon 7); 5'-FAM-CCAGGACCGGCACAAACA (in the p53 gene of normal exon 8), 5'-FAM-CCAGGACAGGCACAAACA (in the p53 gene of mutant exon 8); 5'-FAM-AAGACTTAATACCTGAAGGGTGA (in the p53 gene of normal exon 9) 5'-FAM-AAGACTTAGTACCTGAAGGGTGA (in the p53 gene of mutant exon 9).22

Preparation of the polymers

The dynamic coating matrix of the microchip was made by dissolving 4 mg/mL polyvinylpyrrolidone (PVP, Mr 1 000 000, Sigma Chemicals, St. Louis, MO, USA) in a 1×TBE buffer. The mixture was shaken for 2 min and left to stand for 2 h to remove bubbles. The sieving matrix was prepared by dissolving polyethyle-neoxide (PEO, Mr 8 000 000, Sigma Chemicals, St. Louis, MO, USA) in a 1×TBE buffer with slow stirring overnight.

Microchip electrophoresis procedures

For DNA separation with the polymers as a separa-tion medium, the inner wall of the channel was cova-lently coated with PVP.23 Briefly, PVP solution was manually injected through reservoir 1 to fill the chip channels using a 1 mL medical syringe. Before the separation process, the chip channels were rinsed with 0.1 mol/L HCl, and then flushed by water. The separa-tion medium was filled into the channels by using a 1 mL gas-tight syringe. The sample was pipetted into sample inlet reservoir of the microchip and then was added to the injection cross region using a conventional electrokinetic injection by applying a potential of 500 V at sample outlet reservoir 4 followed by grounding the sample inlet reservoir 2 for 30 s. The applied electric field ranged from 88 to 353 V/cm at buffer inlet (1) and sample outlet (4). After each run, the microchip channel was rinsed with water followed by the running buffer for 5 min each.

Results and discussion

The principles and process for SSCP

The schematic diagram in Figure 1B illustrated the principles and process for ME-SSCP. Double-stranded DNA fragments migrated to the same positions, regard-less of their sequence content. In contrast, the migration of the single-stranded mutant DNA fragments in poly-mer gels differed significantly from those of sin-gle-stranded wild type DNA fragments. So, the identity of the unknown samples was determined by comparing the migration times of the single-stranded DNA frag-ments.

Effect of appropriate concentration of PEO

The main aim of this study was to develop an SSCP- ME method that can provide sensitive and fast detection and separation in mutant and normal ssDNA. Various parameters such as the sieving gel and electric field were examined.

PEO was widely used as a sieving matrix for the rapid separation of the DNA fragments with shorter mi-gration times in electrophoresis.24 However, the separa-tion of DNA fragments mainly depended on the con-centrations of the PEO.25 Therefore, this study first examined the effect of the PEO concentrations ranging from 0.1% to 0.7% on DNA separation. Figure 2 showed the migration times (Figure 2A) and resolution (Figure 2B) of the mutant and normal ssDNA at differ-ent PEO concentrations. The results showed that 0.5% PEO is more suitable for the rapid separation of DNA fragments in terms of the migration time and resolution of the target base pairs. Moreover, a higher viscosity made separation more difficult and increased the time required to fill or clean the sieving matrix in the micro-chip channel.

Figure 2 Migration time (A) and resolution (B) of mutant and normal exons 7, 8 and 9 of the p53 gene at various PEO concen-trations. ME conditions: running buffer 1×TBE (pH 9.2); coat-ing matrix 4 mg/mL (Mr 1 000 000); sieving matrix, 0.8—5.6 mg/mL PEO (Mr 8 000 000); applied electric field 260 V/cm; electrokinetic injection 500 V for 30 s.

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Effect of electric field strength

According to electrophoresis theory, the migration time and resolution of the DNA fragments were in-versely proportional to the electric field. Furthermore, the migration distance of the DNA was much shorter due to fewer physical DNA/polymer entanglement in-teractions with the polymer solutions at the higher elec-tric field.26 However, high electric fields also had a deleterious effect on the DNA fragments and reduced the total migration time due to an increase in the micro-chip temperature from Joule heating. Increased heat in the microchip could lead to broader peaks, non-repro- ducible migration times, sample decomposition, which caused electrical discontinuity through out the channel and decrease in resolving power and efficiency. Using a relatively high electric field, DNA molecules could be separated within a shorter time.27 Therefore, determin-ing the optimum electric field strength was the best ap-proach to ensure the rapid detection of mutant p53 gene in a ME without losing the resolving power.

In order to choose an appropriate electric field, we considered three parameters: resolution, separation speed and read length. While a higher electric field in-creased the resolution and separation speed, it also de-creased the read length. Migration time and peak width were the two parameters affecting resolution. They had the following relationship: R＝2(t2－t1)/w1＋w2,28 where R, t and w were resolution, migration time, and peak width, respectively. Hence, the resolution of mu-tant and normal ssDNA at exons 7, 8 and 9 of p53 gene under the various electric field strength 88, 176, 264 and 353 V/cm was investigated. As shown in Figure 3, the resolution decreased with increasing electric field. Un-der the electric field strength 88 V/cm, the resolution of mutant and normal exon 7 was about 7.8. At the electric field strength 353 V/cm, the resolution of mutant and

Figure 3 Resolution of mutant and normal exons 7, 8 and 9 of the p53 gene under different applied electric fields: 88, 176, 260 and 353 V/cm. ME conditions: running buffer 1×TBE (pH 9.2); coating matrix 4 mg/mL (Mr 1 000 000); sieving matrix, 4 mg/mL PEO (Mr 8 000 000); electrokinetic injection conditions are the same as in Figure 2.

normal exon 7 was about 0.97. On the other hand, the migration time decreased with increasing electric field. For example, the migration time of mutant exon 7 was decreasing from 45 s to 34 s with increasing the electric field from 88 to 260 V/cm. The migration time was shorter when 353 V/cm was used, but the resolution of mutant and normal ssDNA decreased. For compromise, we chose a moderately high electric field of 260 V/cm. Under the optimized conditions, the separation of nor-mal and mutate ssDNA at exons 7, 8 and 9 of the p53 gene with polymer consisting of 4 mg/mL PEO yielded the best resolution, which was shown in Figure 4 A—C.

Performance of the system

The migration time of the mutant and normal ssDNA was the only criterion for identifying the length of indi-vidual fragments in microchip electrophoresis separa-tion. However, drifts in migration time were often ob-served during the separations of a series of samples us-ing the same separation channel. Even in commercial-ized systems, internal standard DNA markers were often required for calibrating the migration times. The repro-ducibility of the migration time during extended usage was therefore an important criterion for evaluating the effectiveness of the method. In this work, a detailed study on the variations in electrophoresis performance was conducted. The results are summarized in Table 1. The precision and absolute values of the migration times remained relatively constant at least within the 3-day study using the same chip, with the precisions of 2.15%—5.81% RSD (n＝5) for the migration times. Theoretical plates of mutant and normal exons 7—9 of the p53 gene obtained for an average of five runs are also shown in Table 1. It should be noted that the above performance was rapidly degraded when the channels were filled with water or a buffer solution without the sieving matrix for any extended periods of over a few hours, obviously because of the breaking of the sorption equilibrium built on the channel walls, in which case new static adsorption treatments would be required to regain the performance.

Table 1 Migration time and theoretical plates of mutant and normal exons 7, 8 and 9 of the p53 gene

Migration time/s (RSD/%) (n＝5)

Theoretical

plate/(103 m－1)

normal mutant normal mutant

Exon 7 29 (3.81) 34 (2.94) 0.59 1.06

Exon 8 25 (5.42) 31 (3.49) 0.57 0.75

Exon 9 37 (5.81) 55 (2.51) 1.31 2.97

Conclusion

In conclusion, we showed an effective method of SSCP-ME with LIF detection for the rapid analysis of mutations at exons 7, 8 and 9 of the p53 gene. The mu-tant and normal ssDNA were separated within 60 s,

Multiplexed p53 Mutation Detection by Microchip Electrophoresis

Chin. J. Chem. 2010, 28, 797—802 © 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 801

Figure 4 Representative ME electropherograms of mutant and normal exons 7 (A) exon 8 (B) and exon 9 (C) of the p53 gene under the optimized conditions. ME-LIF conditions: electroki-netic injection, 500 V for 30 s; separation conditions: applied electric field 260 V/cm; running buffer, 1×TBE (pH 9.2); coat-ing matrix, 4 mg/mL (Mr 1 000 000); sieving matrix, 4 mg/mL PEO (Mr 8 000 000).

which was much faster than that with previously re-ported methods.10,11,29,30 And the labor involved was minimum, which was easier to the reference.31,32 The separation on microfluidic devices is promising; further optimization of sample cleanup and injection methods is underway and could find many clinical and research applications. It is also promising that the present method is applicable to other areas where the qualitative analy-sis or a high resolving power of polymorphism and mu-

tations are required.

Acknowledgements

We are grateful to Shanghai Spectrum Company for the help on the fabrication of LIF detection.

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