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Precise Prediction of DNA Sizes with Transient Isotachophoresis-Capillary Gel Electrophoresis Analysis on a Microchip
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 38, Issue 1, January 2010 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2010, 38(1), 15–20.
RESEARCH PAPER
Precise Prediction of DNA Sizes with Transient Isotachophoresis-Capillary Gel Electrophoresis Analysis on a Microchip LIU Da-Yu1,*, LIANG Guang-Tie1, MO Jian-Kun2, ZHOU Xiao-Mian1 1
Laboratory of Advanced Clinical Chemical Technology, Guangzhou First Municipal People’s Hospital, Affiliated Hospital of Guangzhou Medical College, Guangzhou 510180, China 2 Department of Laboratory Medicine, Guangdong No.2 Provincial People’s Hospital, Guangzhou 510317, China
Abstract: The migration time in transient isotachophoresis (tITP) separation is affected by sample salinity due to the dependence of ITP time on sample-zone conductivity. The sample-to-sample variation of migration time in microchip tITP-capillary gel electrophoresis (CGE) analysis is an undesired factor for precise DNA sizing. In this study, a DNA-sizing method based on relative migration-time proportion (RMP) was developed to eliminate the effect of sample salinity on sizing precision. RMP is defined as the ratio of the migration-time difference between the target fragment and the lower marker to that of the migration-time difference between the upper marker and the lower marker. The RMP values were tested to be reproducible in microchip tITP-CGE separations irrespective of sample salinity. Size of a target DNA was predicted by placing its RMP value in the equation derived from RMPs of standard DNA ladder vs. DNA sizes. The precision and reproducibility of the sizing method were validated by testing multiple standard PCR amplicons. Experimental results showed that the RMP method was simple and reliable, thus well suited to precise DNA sizing with microchip tITP-CGE technique. Key Words:
1
Microchip electrophoresis; Transient isotachophoresis; DNA sizing; Migration time; Sample salinity
Introduction
Microchip electrophoresis (MCE) has become a powerful tool for bio-analysis with advantages of rapidity, high performance, flexible design, low sample and reagent consumption, and so on[1,2]. DNA analysis is one of the most successful applications of MCE. Various kinds of DNA samples have been efficiently analyzed by using MCE[3–5]. The small channel dimension of the microchip facilitates rapid separation and high resolving power; however, the detection sensitivity of the microchip is usually limited due to the low sample loading amounts. Many techniques have been developed to solve the problem of low detection sensitivity in terms of concentration in MCE[6]. One of these techniques is to develop the on-chip sample stacking methods, which appears to
be cost-effective and provides high enrichment effects. Isotachophoresis (ITP) is an effective technique for on-chip DNA preconcentration[7–9], and transient isotachophoresis (tITP) is a simplified ITP scheme for coupling of preconcentration and separation[10]. With ITP or tITP method, the detection sensitivity can be improved without loss of resolution. In tITP, a large volume of sample and leading/terminating ions are injected to the background electrolyte (BGE). By applying current, analytes in the sample zone are stacked into narrow bands one after another according to their mobilities, and at a certain time, separation proceeds by zone electrophoresis in the same channel. It has been reported that highly saline DNA samples could be preconcentrated and separated with simplified microchip tITP-CGE methods[11,12]. The comparison of the migration time of a DNA fragment in
Received 26 July 2009; accepted 6 October 2009 * Corresponding author. Email: [email protected] This work was supported by the National key S&T special project of China (No. 2008ZX10004-004), the National Natural Science Foundation of China (No. 30870753) and the Science and Technology Plan Projects of Science and Technology Bureau of Guangdong Province of China (No.2008B060600036).Copyright © 2010, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(09)60015-3
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
a sample with that of a standard sample analyzed under the same conditions is a general practice for DNA size prediction. However, the migration time of an analyte shows a strong dependence on the sample matrix conditions in tITP analysis. The tITP preconcentration is largely determined by the tITP time, which depends on sample salinity, length of the sample zone, and mobilities of the leading ions, sample ions, and terminating ions[13]. The shift of migration time within samples of different salinity is an undesired factor for peak identification[14]. It has been reported that the migration times of the analytes become predictable irrespective of the matrix salinity by introducing inner standards[14]. In this work, a pair of inner standards, the lower marker and the upper marker were introduced to the tITP-CGE analysis. The relative migration time proportion (RMP), which was determined by the migration time difference of the target DNA fragment and the lower marker as well as that of the upper marker and the lower marker, was used for DNA size prediction. We demonstrated precise and reproducible DNA sizing with the RMP-based method in microchip tITP-CGE analysis. The RMP method can effectively eliminate the drift of migration time caused by difference in sample salinity; thus, it is well suited to precise prediction of DNA size in microchip tITP-CGE analysis.
2
Experimental
2.1
Instruments and reagents
The microchip experiments were performed on a microfluidic chip analyzer with a confocal laser-induced fluorescence (LIF) detector[11]. In brief, the chip analyzer has four individually controlled high-voltage modules to provide sequential voltage output. Data acquisition and processing are carried out using an analog-to-digital (A/D) converter. The detection system and the high-voltage system are synchronized by the operation software. The operation software also displays current, voltage, and electropherogram, and also identifies peaks and calculates migration times, half-height peak widths, peak areas, and heights. Imidazole and (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma (St. Louis, MO, USA). Hydroxypropylmethyl cellulose (HPMC, 40–60 cP, 2% in H2O) was acquired from Aldrich (Milwaukee, WI, USA). The standard PCR amplicons and 100–600 bp DNA ladder were purchased from Tiangen (Beijing, China), and the 50–1500 bp DNA ladder from Takara (Dalian, China). The intercalating DNA dye GeneFinderTM (SYBR Green I) was purchased from Biovision (Xiamen, China) as a 10000 × stock solution. 2.2
The glass plates (6.5 cm × 6.5 cm) with positive photoresist for microchip fabrication were from Shaoguang (Changsha, China). The glass microchip was fabricated with standard lithography, wet-etching, and thermo-bonding method as previously described[15]. Sample injection segment of the double-T structure was 7.5 mm, and the effective separation distance was 4 cm. All the channels were 30 μm deep and 50 μm wide at half-height. The micro-channel was coated with linear polyacrylamide using the method developed by Hjertén[16]. 2.2.2
The background buffer for microchip tITP-CGE was 20 mM HEPES/40 mM imidazole at pH 7.5. The running buffer contained various concentrations of HPMC-50 as sieving matrix and 1 × GeneFinder as the intercalating dye. All buffers were prepared with double distilled water (ddH2O). Before each run, the running buffer was loaded to the reservoirs defined as buffer waste (BW), buffer (B), and sample waste (SW) (Fig.1). By addressing the negative pressure with a vacuum pump at the sample reservoir (S), all channels were filled with the sieving buffer. Finally, the sample was loaded to S. The microchip tITP-CGE analysis included two steps, sample injection and separation. Sample injection was performed by applying a potential (300 V cm–1) across the S and SW, with S at ground for 30 s. For separation, S and SW were floating and high voltage was applied to the BW with ground at the B for 300 s.
3
Results and discussion
3.1
Principle of tITP-CGE method
The sample matrix of DNA usually contains high concentrations of salt. On account of the dependence of sample injection on the conductivity of the sample buffer, the highly saline buffer reduces the amount of DNA that is electrokinetically loaded[17], and thus the detection sensitivity of DNA sample with the MCE analysis is usually limited.
Methods Fig.1
2.2.1
Microchip tITP-CGE
Microchip Fabrication
Illustration of tITP-CGE analysis on a microchip with double-T sample injector unit
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
The on-chip tITP-CGE analysis can effectively improve the detection sensitivity of the highly saline DNA samples by increasing sample loading volume. In the tITP-CGE analysis, HEPES and chloride ions were selected as terminating and leading ions, respectively. DNA fragments have an intermediate mobility that is sandwiched between chloride and HEPES. Chloride ions in the sample matrix can potentially act as the leading ions. Therefore, the microchip tITP-CGE analysis provides a simplified scheme for preconcentration and separation of DNA samples. As shown in Fig.1, the microchip tITP-CGE analysis includes sample loading, tITP preconcentration, and CGE separation. The double-T structured sample injector enables increased sample loading volume compared with crossshaped microchip. With the application of electrical current, the chloride ions will quickly leave the dsDNA to form a leading zone. The chloride, dsDNA, and HEPES ions are ordered according to decreasing mobilities. The electric field strength increases from leading to terminating zone. A conductivity discontinuity will be formed behind the rear boundary of the leading zone, resulting in nonuniformity of the electric field strength. As a result of the high electric field strength applied behind the leading zone, the dsDNA fragments are stacked into narrow bands one after another. The leading zone will broaden during migration due to electromigration dispersion. The concentration of the higher mobility chloride ions decreases with time, leading to decreasing differences in the electric field strength along the migrating zones. At a certain concentration of the leading zone, the dsDNAs destack one after another according to their mobilities and move in CGE mode. With this technique, the coupling of ITP preconcentration to CGE separation on a microchip can improve detection sensitivity without loss of resolution. The operation can be carried out with single running buffer and four electrodes, which is as simple as that of a standard CGE. 3.2
Effects of sample salinity on migration time in tITP-CGE analysis
The tITP preconcentration is largely determined by sample salinity. The concentration of sample in the stacked plug is regulated by the leading ions according to the Kohlrausch regulating function (KRF)[18]; on the other side, the tITP time also depends on sample salinity[13]. In the tITP-CGE analysis, the migration time tM to the detector point can be obtained from the sum of the ITP-time and the CGE-time to the detector point of one analyte. tM = tITP + tCGE (1) As tITP is a sample-zone sharping process without separation, the tITP-time affects the starting point of CGE and therefore, the migration times of DNA fragments. For sample i, in a sample matrix containing leading ions,
the ITP-time can be estimated by tITP = lsțs(ȝL – ȝT)/[I((ȝL – ȝi)2] (2)[13] Where, ls and țs are length and conductivity of the sample zone, respectively. I is the current, ȝL, ȝT and ȝi are mobilities of the leading ions, sample ions and terminating ions, respectively. ȝL >ȝi > ȝT. The conductivity of the sample zone țs = F•ȈCiZiUi (3) where, F is the Faraday constant, Ci is the concentration of the matrix ions, Ui is the absolute electrophoretic mobilities of the matrix ionˈand Zi is the charge of matrix ion. It is obvious from these equations that the migration time is influenced by sample salinity. A shortening of the migration time will be obtained for low sample salinities, and in contrast, high sample salinities lead to a longer migration time and the delay of CGE start. As shown in Fig.2, there was an obvious shift in migration time between the 200 bp PCR amplicon and DNA marker of the same size due to the difference in sample salinities. The shift of migration time in tITP-CGE analysis was a hindrance for precise DNA sizing. Therefore, it is necessary to develop a method for accurate prediction of the DNA size in the microchip tITP-CGE analysis. 3.3
RMP-based DNA-sizing approach
The aim of the RMP method is to eliminate the drift of migration time in tITP-CGE analysis by introducing relative migration-time proportion (RMP) as a parameter for DNA sizing. Sample salinity affects the migration time but not the RMP, which depends only on the length of DNA fragment. The RMP-sizing method is illustrated in Fig.3. For a separation with two internal standards that bracket all the target DNA fragments, the relative migration time ǻT = TU – TL, where TL and TU are the migration time of the lower marker and the upper marker, respectively. The TU should be longer, while TL should be shorter than the migration time of all the DNA fragments in the sample. For a target DNA fragment with migration time T, the relative migration time
Fig.2 Microchip tITP-CGE analysis of 200 bp PCR product and 100–600 bp DNA ladder The sample matrix of the DNA ladder was TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA) and that of the PCR product was PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2)
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
ǻt = T – TL. The RMP value is defined as ¨t/¨T. With this method, the lower marker has an RMP value of 0, while the upper marker has an RMP value of 1. By analyzing the standard DNA ladder against the dual internal standards, the plots can be derived from RMPs vs. DNA sizes. The resulted plots can be arranged in the exponential decay model to get the fitting equation. The target DNA fragment can be analyzed against the same dual internal standards to get its RMP value. The size of a DNA fragment can be calculated by fitting its RMP value to the equation. As shown in Fig.3, nearly all the plots fell on the curve, indicating a good correlation between RMP and DNA size. It was shown in Table 1 that the average correlation coefficients (R2) of the fitting equation for 10 individual separations were 0.9998 and 0.9905, corresponding to DNA-sizing ranges of 16–600 bp and 50–1500 bp (Table 1). Average RSDs of RMP were less than 3%, indicating satisfactory reproducibility of RMP. These results indicated that RMP was able to provide accurate sizing information. Moreover, since all the curves start at 0 and end at 1, separations with a certain pair of internal standards could be aligned by RMP to eliminate the drift of migration time. The effects of electric field strength, sample matrix, and sieving polymer concentration on sizing accuracy were also investigated (Table 2). The fitting equation for DNA sizing was set up by analyzing a standard DNA ladder against the dual
internal standards. Sizing precision was evaluated with the error percentage of the predicted DNA sizes. The lowest average sizing error was found under the separation electric field strength of 190 V cm–1, while the highest under 150 V cm–1, indicating that the lower electric field strength was an adverse factor for sizing accuracy. The poor sizing precision under low separation electric field strength can be ascribed to lower preconcentration rates, which cause longer tITP time and peak broadening that are disadvantageous for accurate identification of migration time. Here, the separation electric field strength of 190 V cm–1 was selected in the following analyses. Pertaining to the dependence of sample injection on the conductivity of the sample buffer and the electrophoresis buffer[19], the high ionic strength sample matrix significantly reduced the signal intensity. However, it was found that reproducible RMP and sizing accuracy was obtained with different sample matrix (Fig.4). Changes in HPMC concentration apparently affected the migration time, but the sizing precision was relatively stable. However, it should be noted that the RMP value of a DNA fragment changed with HPMC concentration as sieving polymer of different concentration has selective resolution between peaks. Thus, the calibration curve derived from RMPs vs. DNA sizes is only applicable to separations with the same running buffer. These results showed that the effect of sample salinity on DNA-sizing precision could be eliminated by using RMP methods in a wide range of experimental conditions.
Fig.3 Illustration of RMP DNA-sizing approach Table 1 Reproducibility of RMP and its correlation with DNA size Separation condition
Average RSD of RMP Values (%, n = 10)
Internal Standards
Sample Matrix
100 bp
200 bp
300 bp
400 bp
500 bp
600 bp
800 bp
1000 bp
Average R2 of fitting equation
16–600 bp *a
TE PCR
1.6 1.8
1.8 1.8
1.7 1.7
2.0 1.9
2.2 2.1
ņ ņ
ņ ņ
ņ ņ
0.9998 0.9997
16–800 bp *b 50–1500 bp *c
TE PCR TE PCR
0.5 0.6 0.3 0.4
0.5 0.5 0.7 0.7
0.3 0.3 0.9 0.8
1.0 0.9 1.3 1.3
0.1 0.2 1.5 1.5
2.5 2.4 2.0 2.0
ņ ņ 2.0 1.9
ņ ņ 2.8 2.6
0.9985 0.9987 0.9905 0.9921
*a. HPMC concentration 2%; *b. HPMC concentration 1.5%; *c. HPMC concentration 0.8%. TE buffer: 10 Mm Tris-HCl, 1 Mm EDTA, PCR buffer: 10 mM Tris-HCl, 50 mmol/L KCl, 2.5 mmol/L, MgCl2. The analytical data were from tITP-CGE analysis on a microchip
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
Table 2 Average errors (%) of predicted sizes under different separation conditions Average errors of predicted sizes (%) DNA (bp)
Electric Field Strength (V cm–1) 0.5 × PCR buffer 0.8% HPMC
HPMC concentration 190 V cm–1 Field Strength 0.5 × PCR buffer
Salinity of sample matrix 190 V cm–1 Field Strength 0.8% HPMC
210
190
170
150
10 mM KCl
35 mM KCl
70 mM KCl
0.8%*a
1.5%*b
2%*c
100 200 300 400 500 600 700 800 900 1000
2.5 0.7 3.0 6.9 2.7 1.3 5.4 0.3 1.1 2.5
1.5 2.2 0.5 3.5 0.3 0.9 1.9 1.9 0.2 0.8
0.4 5.3 3.8 0.4 4.2 5.3 2.3 6.0 4.4 2.1
3.5 10.1 9.7 6.3 8.9 9.2 8.3 10.4 9.6 5.5
0.5 2.2 0.4 4.2 0.0 1.0 4.1 1.6 0.4 1.3
1.5 2.2 0.5 3.5 0.3 0.9 1.9 2.0 0.2 0.8
2.1 1.3 0.9 4.9 0.7 0.3 3.4 1.5 0.2 1.2
1.5 2.2 0.5 3.5 0.3 0.9 1.9 2.0 0.2 0.8
3.0 4.7 2.4 2.9 0.8 0.2 ņ ņ ņ ņ
4.3 2.0 1.7 0.2 0.5 ņ ņ ņ ņ ņ
Average
2.63
1.36
3.42
8.16
1.57
1.36
1.64
1.36
2.33
1.72
The figures are average values from 10 parallel separations. *a. lower-marker 50 bp, upper-marker 1500 bp; *b. lower-marker 16 bp, upper-marker 800 bp; *c. lower-marker 16 bp, upper-marker 600 bp.
Fig.4 Waterfall mode electropherograms showing DNA samples of different salinity that were analyzed with microchip tITP-CGE (a), and electropherograms aligned by RMP (b) The sizes of DNA fragments analyzed were 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 bp
3.4
Prediction of DNA sizes with tITP-CGE analysis on a microchip
The RMP-sizing method was tested by consecutive microchip tITP-CGE analysis (Fig.5), with which the sizing precision was investigated with individual separations. The standard PCR samples were analyzed with 16–600 bp or 50–1500 bp internal standards. With the RMP approach, the sample-to-sample variations in migration time could be virtually eliminated. The RMP-sizing assay showed satisfied accuracies. As shown in Table 3, the average sizing error was 1.32% within 16–600 bp and 1.04% within 50–1500 bp. As precision of dsDNA sizing is affected by the conformation of DNA strand as well as the composition of nucleotide base[19,20], the size prediction is not expected to be as accurate as that of single-stranded (ssDNA) separation. Sizing precision using RMP method in our analyses is comparable to that reported previously[21]. Our experimental results showed that RMP method could eliminate the influence of irreproducible migration time on DNA sizing, and was suited to precise DNA sizing with microchip tITP-CGE analysis.
4
Conclusions
We have developed an RMP-based method for simple DNA sizing with on-chip tITP-CGE analysis. Experimental results showed that the RMP method can effectively eliminate the drift of migration time caused by the difference in sample salinity. The current method is simple and reliable, thus well suited for precise DNA sizing with microchip tITP-CGE analysis.
Fig.5 Analytical data obtained from microchip tITP-CGE analysis Sample matrix of the DNA ladder was 10 mM Tris-HCl, 1 mM EDTA, while that of the PCR products was 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2. Lower-marker: 16 bp, Uppermarker: 600 bp. Numbers on electropherograms correspond to the sizes of PCR product in bp
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
Table 3 Predicted sizes of the standard PCR amplicons using RMP method DNA fragment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Average
DNA sizing within 16–600 bp*a Actual Size (bp) Predicted Size (bp) 100 102 200 196 300 305 400 400 500 498 — — — — — — — — — — — — — — — — — —
Error (%) 2.3 2.0 1.7 0.1 0.5 — — — — — — — — — 1.32
Actual Size (bp) 100 150 200 250 300 350 400 450 500 600 700 800 900 1000
DNA sizing within 50–1500 bp*b Predicted Size (bp) Error (%) 101 1.0 150 0.0 196 2.0 248 0.8 299 0.3 350 0.0 414 3.5 446 0.9 498 0.4 595 0.8 713 1.9 784 2.0 898 0.2 1008 0.8 1.04
*a. Separation conditions: 1.5 % HPMC, 190 V/cm electric field strength, 16 bp lower-marker and 600 bp upper-marker; *b. Separation conditions: 0.8% HPMC, 190 V/cm electric field strength, 50 bp lower-marker and 1500 bp upper-marker
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Cite this article as: Chin J Anal Chem, 2010, 38(1), 15–20.
RESEARCH PAPER
Precise Prediction of DNA Sizes with Transient Isotachophoresis-Capillary Gel Electrophoresis Analysis on a Microchip LIU Da-Yu1,*, LIANG Guang-Tie1, MO Jian-Kun2, ZHOU Xiao-Mian1 1
Laboratory of Advanced Clinical Chemical Technology, Guangzhou First Municipal People’s Hospital, Affiliated Hospital of Guangzhou Medical College, Guangzhou 510180, China 2 Department of Laboratory Medicine, Guangdong No.2 Provincial People’s Hospital, Guangzhou 510317, China
Abstract: The migration time in transient isotachophoresis (tITP) separation is affected by sample salinity due to the dependence of ITP time on sample-zone conductivity. The sample-to-sample variation of migration time in microchip tITP-capillary gel electrophoresis (CGE) analysis is an undesired factor for precise DNA sizing. In this study, a DNA-sizing method based on relative migration-time proportion (RMP) was developed to eliminate the effect of sample salinity on sizing precision. RMP is defined as the ratio of the migration-time difference between the target fragment and the lower marker to that of the migration-time difference between the upper marker and the lower marker. The RMP values were tested to be reproducible in microchip tITP-CGE separations irrespective of sample salinity. Size of a target DNA was predicted by placing its RMP value in the equation derived from RMPs of standard DNA ladder vs. DNA sizes. The precision and reproducibility of the sizing method were validated by testing multiple standard PCR amplicons. Experimental results showed that the RMP method was simple and reliable, thus well suited to precise DNA sizing with microchip tITP-CGE technique. Key Words:
1
Microchip electrophoresis; Transient isotachophoresis; DNA sizing; Migration time; Sample salinity
Introduction
Microchip electrophoresis (MCE) has become a powerful tool for bio-analysis with advantages of rapidity, high performance, flexible design, low sample and reagent consumption, and so on[1,2]. DNA analysis is one of the most successful applications of MCE. Various kinds of DNA samples have been efficiently analyzed by using MCE[3–5]. The small channel dimension of the microchip facilitates rapid separation and high resolving power; however, the detection sensitivity of the microchip is usually limited due to the low sample loading amounts. Many techniques have been developed to solve the problem of low detection sensitivity in terms of concentration in MCE[6]. One of these techniques is to develop the on-chip sample stacking methods, which appears to
be cost-effective and provides high enrichment effects. Isotachophoresis (ITP) is an effective technique for on-chip DNA preconcentration[7–9], and transient isotachophoresis (tITP) is a simplified ITP scheme for coupling of preconcentration and separation[10]. With ITP or tITP method, the detection sensitivity can be improved without loss of resolution. In tITP, a large volume of sample and leading/terminating ions are injected to the background electrolyte (BGE). By applying current, analytes in the sample zone are stacked into narrow bands one after another according to their mobilities, and at a certain time, separation proceeds by zone electrophoresis in the same channel. It has been reported that highly saline DNA samples could be preconcentrated and separated with simplified microchip tITP-CGE methods[11,12]. The comparison of the migration time of a DNA fragment in
Received 26 July 2009; accepted 6 October 2009 * Corresponding author. Email: [email protected] This work was supported by the National key S&T special project of China (No. 2008ZX10004-004), the National Natural Science Foundation of China (No. 30870753) and the Science and Technology Plan Projects of Science and Technology Bureau of Guangdong Province of China (No.2008B060600036).Copyright © 2010, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(09)60015-3
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
a sample with that of a standard sample analyzed under the same conditions is a general practice for DNA size prediction. However, the migration time of an analyte shows a strong dependence on the sample matrix conditions in tITP analysis. The tITP preconcentration is largely determined by the tITP time, which depends on sample salinity, length of the sample zone, and mobilities of the leading ions, sample ions, and terminating ions[13]. The shift of migration time within samples of different salinity is an undesired factor for peak identification[14]. It has been reported that the migration times of the analytes become predictable irrespective of the matrix salinity by introducing inner standards[14]. In this work, a pair of inner standards, the lower marker and the upper marker were introduced to the tITP-CGE analysis. The relative migration time proportion (RMP), which was determined by the migration time difference of the target DNA fragment and the lower marker as well as that of the upper marker and the lower marker, was used for DNA size prediction. We demonstrated precise and reproducible DNA sizing with the RMP-based method in microchip tITP-CGE analysis. The RMP method can effectively eliminate the drift of migration time caused by difference in sample salinity; thus, it is well suited to precise prediction of DNA size in microchip tITP-CGE analysis.
2
Experimental
2.1
Instruments and reagents
The microchip experiments were performed on a microfluidic chip analyzer with a confocal laser-induced fluorescence (LIF) detector[11]. In brief, the chip analyzer has four individually controlled high-voltage modules to provide sequential voltage output. Data acquisition and processing are carried out using an analog-to-digital (A/D) converter. The detection system and the high-voltage system are synchronized by the operation software. The operation software also displays current, voltage, and electropherogram, and also identifies peaks and calculates migration times, half-height peak widths, peak areas, and heights. Imidazole and (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma (St. Louis, MO, USA). Hydroxypropylmethyl cellulose (HPMC, 40–60 cP, 2% in H2O) was acquired from Aldrich (Milwaukee, WI, USA). The standard PCR amplicons and 100–600 bp DNA ladder were purchased from Tiangen (Beijing, China), and the 50–1500 bp DNA ladder from Takara (Dalian, China). The intercalating DNA dye GeneFinderTM (SYBR Green I) was purchased from Biovision (Xiamen, China) as a 10000 × stock solution. 2.2
The glass plates (6.5 cm × 6.5 cm) with positive photoresist for microchip fabrication were from Shaoguang (Changsha, China). The glass microchip was fabricated with standard lithography, wet-etching, and thermo-bonding method as previously described[15]. Sample injection segment of the double-T structure was 7.5 mm, and the effective separation distance was 4 cm. All the channels were 30 μm deep and 50 μm wide at half-height. The micro-channel was coated with linear polyacrylamide using the method developed by Hjertén[16]. 2.2.2
The background buffer for microchip tITP-CGE was 20 mM HEPES/40 mM imidazole at pH 7.5. The running buffer contained various concentrations of HPMC-50 as sieving matrix and 1 × GeneFinder as the intercalating dye. All buffers were prepared with double distilled water (ddH2O). Before each run, the running buffer was loaded to the reservoirs defined as buffer waste (BW), buffer (B), and sample waste (SW) (Fig.1). By addressing the negative pressure with a vacuum pump at the sample reservoir (S), all channels were filled with the sieving buffer. Finally, the sample was loaded to S. The microchip tITP-CGE analysis included two steps, sample injection and separation. Sample injection was performed by applying a potential (300 V cm–1) across the S and SW, with S at ground for 30 s. For separation, S and SW were floating and high voltage was applied to the BW with ground at the B for 300 s.
3
Results and discussion
3.1
Principle of tITP-CGE method
The sample matrix of DNA usually contains high concentrations of salt. On account of the dependence of sample injection on the conductivity of the sample buffer, the highly saline buffer reduces the amount of DNA that is electrokinetically loaded[17], and thus the detection sensitivity of DNA sample with the MCE analysis is usually limited.
Methods Fig.1
2.2.1
Microchip tITP-CGE
Microchip Fabrication
Illustration of tITP-CGE analysis on a microchip with double-T sample injector unit
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
The on-chip tITP-CGE analysis can effectively improve the detection sensitivity of the highly saline DNA samples by increasing sample loading volume. In the tITP-CGE analysis, HEPES and chloride ions were selected as terminating and leading ions, respectively. DNA fragments have an intermediate mobility that is sandwiched between chloride and HEPES. Chloride ions in the sample matrix can potentially act as the leading ions. Therefore, the microchip tITP-CGE analysis provides a simplified scheme for preconcentration and separation of DNA samples. As shown in Fig.1, the microchip tITP-CGE analysis includes sample loading, tITP preconcentration, and CGE separation. The double-T structured sample injector enables increased sample loading volume compared with crossshaped microchip. With the application of electrical current, the chloride ions will quickly leave the dsDNA to form a leading zone. The chloride, dsDNA, and HEPES ions are ordered according to decreasing mobilities. The electric field strength increases from leading to terminating zone. A conductivity discontinuity will be formed behind the rear boundary of the leading zone, resulting in nonuniformity of the electric field strength. As a result of the high electric field strength applied behind the leading zone, the dsDNA fragments are stacked into narrow bands one after another. The leading zone will broaden during migration due to electromigration dispersion. The concentration of the higher mobility chloride ions decreases with time, leading to decreasing differences in the electric field strength along the migrating zones. At a certain concentration of the leading zone, the dsDNAs destack one after another according to their mobilities and move in CGE mode. With this technique, the coupling of ITP preconcentration to CGE separation on a microchip can improve detection sensitivity without loss of resolution. The operation can be carried out with single running buffer and four electrodes, which is as simple as that of a standard CGE. 3.2
Effects of sample salinity on migration time in tITP-CGE analysis
The tITP preconcentration is largely determined by sample salinity. The concentration of sample in the stacked plug is regulated by the leading ions according to the Kohlrausch regulating function (KRF)[18]; on the other side, the tITP time also depends on sample salinity[13]. In the tITP-CGE analysis, the migration time tM to the detector point can be obtained from the sum of the ITP-time and the CGE-time to the detector point of one analyte. tM = tITP + tCGE (1) As tITP is a sample-zone sharping process without separation, the tITP-time affects the starting point of CGE and therefore, the migration times of DNA fragments. For sample i, in a sample matrix containing leading ions,
the ITP-time can be estimated by tITP = lsțs(ȝL – ȝT)/[I((ȝL – ȝi)2] (2)[13] Where, ls and țs are length and conductivity of the sample zone, respectively. I is the current, ȝL, ȝT and ȝi are mobilities of the leading ions, sample ions and terminating ions, respectively. ȝL >ȝi > ȝT. The conductivity of the sample zone țs = F•ȈCiZiUi (3) where, F is the Faraday constant, Ci is the concentration of the matrix ions, Ui is the absolute electrophoretic mobilities of the matrix ionˈand Zi is the charge of matrix ion. It is obvious from these equations that the migration time is influenced by sample salinity. A shortening of the migration time will be obtained for low sample salinities, and in contrast, high sample salinities lead to a longer migration time and the delay of CGE start. As shown in Fig.2, there was an obvious shift in migration time between the 200 bp PCR amplicon and DNA marker of the same size due to the difference in sample salinities. The shift of migration time in tITP-CGE analysis was a hindrance for precise DNA sizing. Therefore, it is necessary to develop a method for accurate prediction of the DNA size in the microchip tITP-CGE analysis. 3.3
RMP-based DNA-sizing approach
The aim of the RMP method is to eliminate the drift of migration time in tITP-CGE analysis by introducing relative migration-time proportion (RMP) as a parameter for DNA sizing. Sample salinity affects the migration time but not the RMP, which depends only on the length of DNA fragment. The RMP-sizing method is illustrated in Fig.3. For a separation with two internal standards that bracket all the target DNA fragments, the relative migration time ǻT = TU – TL, where TL and TU are the migration time of the lower marker and the upper marker, respectively. The TU should be longer, while TL should be shorter than the migration time of all the DNA fragments in the sample. For a target DNA fragment with migration time T, the relative migration time
Fig.2 Microchip tITP-CGE analysis of 200 bp PCR product and 100–600 bp DNA ladder The sample matrix of the DNA ladder was TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA) and that of the PCR product was PCR buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2)
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
ǻt = T – TL. The RMP value is defined as ¨t/¨T. With this method, the lower marker has an RMP value of 0, while the upper marker has an RMP value of 1. By analyzing the standard DNA ladder against the dual internal standards, the plots can be derived from RMPs vs. DNA sizes. The resulted plots can be arranged in the exponential decay model to get the fitting equation. The target DNA fragment can be analyzed against the same dual internal standards to get its RMP value. The size of a DNA fragment can be calculated by fitting its RMP value to the equation. As shown in Fig.3, nearly all the plots fell on the curve, indicating a good correlation between RMP and DNA size. It was shown in Table 1 that the average correlation coefficients (R2) of the fitting equation for 10 individual separations were 0.9998 and 0.9905, corresponding to DNA-sizing ranges of 16–600 bp and 50–1500 bp (Table 1). Average RSDs of RMP were less than 3%, indicating satisfactory reproducibility of RMP. These results indicated that RMP was able to provide accurate sizing information. Moreover, since all the curves start at 0 and end at 1, separations with a certain pair of internal standards could be aligned by RMP to eliminate the drift of migration time. The effects of electric field strength, sample matrix, and sieving polymer concentration on sizing accuracy were also investigated (Table 2). The fitting equation for DNA sizing was set up by analyzing a standard DNA ladder against the dual
internal standards. Sizing precision was evaluated with the error percentage of the predicted DNA sizes. The lowest average sizing error was found under the separation electric field strength of 190 V cm–1, while the highest under 150 V cm–1, indicating that the lower electric field strength was an adverse factor for sizing accuracy. The poor sizing precision under low separation electric field strength can be ascribed to lower preconcentration rates, which cause longer tITP time and peak broadening that are disadvantageous for accurate identification of migration time. Here, the separation electric field strength of 190 V cm–1 was selected in the following analyses. Pertaining to the dependence of sample injection on the conductivity of the sample buffer and the electrophoresis buffer[19], the high ionic strength sample matrix significantly reduced the signal intensity. However, it was found that reproducible RMP and sizing accuracy was obtained with different sample matrix (Fig.4). Changes in HPMC concentration apparently affected the migration time, but the sizing precision was relatively stable. However, it should be noted that the RMP value of a DNA fragment changed with HPMC concentration as sieving polymer of different concentration has selective resolution between peaks. Thus, the calibration curve derived from RMPs vs. DNA sizes is only applicable to separations with the same running buffer. These results showed that the effect of sample salinity on DNA-sizing precision could be eliminated by using RMP methods in a wide range of experimental conditions.
Fig.3 Illustration of RMP DNA-sizing approach Table 1 Reproducibility of RMP and its correlation with DNA size Separation condition
Average RSD of RMP Values (%, n = 10)
Internal Standards
Sample Matrix
100 bp
200 bp
300 bp
400 bp
500 bp
600 bp
800 bp
1000 bp
Average R2 of fitting equation
16–600 bp *a
TE PCR
1.6 1.8
1.8 1.8
1.7 1.7
2.0 1.9
2.2 2.1
ņ ņ
ņ ņ
ņ ņ
0.9998 0.9997
16–800 bp *b 50–1500 bp *c
TE PCR TE PCR
0.5 0.6 0.3 0.4
0.5 0.5 0.7 0.7
0.3 0.3 0.9 0.8
1.0 0.9 1.3 1.3
0.1 0.2 1.5 1.5
2.5 2.4 2.0 2.0
ņ ņ 2.0 1.9
ņ ņ 2.8 2.6
0.9985 0.9987 0.9905 0.9921
*a. HPMC concentration 2%; *b. HPMC concentration 1.5%; *c. HPMC concentration 0.8%. TE buffer: 10 Mm Tris-HCl, 1 Mm EDTA, PCR buffer: 10 mM Tris-HCl, 50 mmol/L KCl, 2.5 mmol/L, MgCl2. The analytical data were from tITP-CGE analysis on a microchip
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
Table 2 Average errors (%) of predicted sizes under different separation conditions Average errors of predicted sizes (%) DNA (bp)
Electric Field Strength (V cm–1) 0.5 × PCR buffer 0.8% HPMC
HPMC concentration 190 V cm–1 Field Strength 0.5 × PCR buffer
Salinity of sample matrix 190 V cm–1 Field Strength 0.8% HPMC
210
190
170
150
10 mM KCl
35 mM KCl
70 mM KCl
0.8%*a
1.5%*b
2%*c
100 200 300 400 500 600 700 800 900 1000
2.5 0.7 3.0 6.9 2.7 1.3 5.4 0.3 1.1 2.5
1.5 2.2 0.5 3.5 0.3 0.9 1.9 1.9 0.2 0.8
0.4 5.3 3.8 0.4 4.2 5.3 2.3 6.0 4.4 2.1
3.5 10.1 9.7 6.3 8.9 9.2 8.3 10.4 9.6 5.5
0.5 2.2 0.4 4.2 0.0 1.0 4.1 1.6 0.4 1.3
1.5 2.2 0.5 3.5 0.3 0.9 1.9 2.0 0.2 0.8
2.1 1.3 0.9 4.9 0.7 0.3 3.4 1.5 0.2 1.2
1.5 2.2 0.5 3.5 0.3 0.9 1.9 2.0 0.2 0.8
3.0 4.7 2.4 2.9 0.8 0.2 ņ ņ ņ ņ
4.3 2.0 1.7 0.2 0.5 ņ ņ ņ ņ ņ
Average
2.63
1.36
3.42
8.16
1.57
1.36
1.64
1.36
2.33
1.72
The figures are average values from 10 parallel separations. *a. lower-marker 50 bp, upper-marker 1500 bp; *b. lower-marker 16 bp, upper-marker 800 bp; *c. lower-marker 16 bp, upper-marker 600 bp.
Fig.4 Waterfall mode electropherograms showing DNA samples of different salinity that were analyzed with microchip tITP-CGE (a), and electropherograms aligned by RMP (b) The sizes of DNA fragments analyzed were 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 bp
3.4
Prediction of DNA sizes with tITP-CGE analysis on a microchip
The RMP-sizing method was tested by consecutive microchip tITP-CGE analysis (Fig.5), with which the sizing precision was investigated with individual separations. The standard PCR samples were analyzed with 16–600 bp or 50–1500 bp internal standards. With the RMP approach, the sample-to-sample variations in migration time could be virtually eliminated. The RMP-sizing assay showed satisfied accuracies. As shown in Table 3, the average sizing error was 1.32% within 16–600 bp and 1.04% within 50–1500 bp. As precision of dsDNA sizing is affected by the conformation of DNA strand as well as the composition of nucleotide base[19,20], the size prediction is not expected to be as accurate as that of single-stranded (ssDNA) separation. Sizing precision using RMP method in our analyses is comparable to that reported previously[21]. Our experimental results showed that RMP method could eliminate the influence of irreproducible migration time on DNA sizing, and was suited to precise DNA sizing with microchip tITP-CGE analysis.
4
Conclusions
We have developed an RMP-based method for simple DNA sizing with on-chip tITP-CGE analysis. Experimental results showed that the RMP method can effectively eliminate the drift of migration time caused by the difference in sample salinity. The current method is simple and reliable, thus well suited for precise DNA sizing with microchip tITP-CGE analysis.
Fig.5 Analytical data obtained from microchip tITP-CGE analysis Sample matrix of the DNA ladder was 10 mM Tris-HCl, 1 mM EDTA, while that of the PCR products was 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2. Lower-marker: 16 bp, Uppermarker: 600 bp. Numbers on electropherograms correspond to the sizes of PCR product in bp
LIU Da-Yu et al. / Chinese Journal of Analytical Chemistry, 2010, 38(1): 15–20
Table 3 Predicted sizes of the standard PCR amplicons using RMP method DNA fragment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Average
DNA sizing within 16–600 bp*a Actual Size (bp) Predicted Size (bp) 100 102 200 196 300 305 400 400 500 498 — — — — — — — — — — — — — — — — — —
Error (%) 2.3 2.0 1.7 0.1 0.5 — — — — — — — — — 1.32
Actual Size (bp) 100 150 200 250 300 350 400 450 500 600 700 800 900 1000
DNA sizing within 50–1500 bp*b Predicted Size (bp) Error (%) 101 1.0 150 0.0 196 2.0 248 0.8 299 0.3 350 0.0 414 3.5 446 0.9 498 0.4 595 0.8 713 1.9 784 2.0 898 0.2 1008 0.8 1.04
*a. Separation conditions: 1.5 % HPMC, 190 V/cm electric field strength, 16 bp lower-marker and 600 bp upper-marker; *b. Separation conditions: 0.8% HPMC, 190 V/cm electric field strength, 50 bp lower-marker and 1500 bp upper-marker
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