6
Journal of Chromatography A, 1156 (2007) 154–159 Miniaturised isotachophoresis of DNA Jeff E. Prest a,, Sara J. Baldock a , Philip J.R. Day b , Peter R. Fielden a , Nicholas J. Goddard a , Bernard J. Treves Brown a a School of Chemical Engineering and Analytical Science, The University of Manchester, P.O. Box 88, Manchester M60 1QD, UK b CIGMR, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK Available online 12 January 2007 Abstract This paper presents the findings of a feasibility study investigating the behaviour of DNA under conditions of miniaturised isotachophoresis. An electrolyte system comprising a leading electrolyte of 5 mM perchloric acid at pH 6.0 and a terminating electrolyte of 10 mM gallic acid was devised and used to perform isotachophoresis of DNA containing samples on a miniaturised poly(methyl methacrylate) device. Under such conditions it was found that no separation of DNA fragments was observed with the substance migrating instead as a single isotachophoretic zone. Whilst such a result shows the method is unsuitable for analysis DNA it offers significant potential as a means of sample preparation for subsequent analysis using another method. This is because the single zone of DNA formed is preconcentrated to a constant concentration governed by the leading ion and is separated from all species with different effective electrophoretic mobilities. © 2007 Elsevier B.V. All rights reserved. Keywords: DNA; Isotachophoresis; Sample preparation; Miniaturisation; Microdevice; Chip 1. Introduction Electrophoretic methods have proven themselves to be use- ful tools in analysing nucleic acids. In particular, the use of capillary zone electrophoresis (CZE) and capillary gel elec- trophoresis (CGE) have been found to be effective methods for the separation of DNA fragments [1,2]. These methods have also proven readily amenable to implementation on a miniaturised scale [3,4]. Miniaturisation offers a range of benefits includ- ing improved analytical performance, reduced analysis times and the relative ease of performing parallel analyses. These lat- ter two features allow for high throughput operations and were a major part of the reason why these electrophoretic methods were widely used in the project to map the human genome [5]. Isotachophoresis (ITP) is another member of the family of electroseparation techniques, albeit one that is somewhat less commonly encountered than CZE. ITP does however offer a number of useful features not found in CZE. The most useful of these is the ability to control separation parameters by vary- Corresponding author. Tel.: +44 161 306 8900; fax: +44 161 306 4896. E-mail address: [email protected] (J.E. Prest). ing the electrolyte system used. The concentration of the leading electrolyte governs the concentration that all of the sample zones adopt, enabling ITP to be used as a method to preconcentrate dilute samples. Like many other electroseparation methods ITP can be readily miniaturised. When performed in such a format the technique has been used as both a separation method in its own right [6,7] or as a sample preparation step prior to perform- ing a CZE separation [8,9]. Miniaturised ITP has been used with a wide variety of samples as evidenced by the range of applica- tions shown in a recent review on the topic [10]. However, to date there have been few reported uses of miniaturised ITP involving DNA samples. The use of the method has seen some use as a pre- concentration method applied either conventionally (ITP–ZE) [11] or as transient ITP (TrITP–CGE) [12,13] for separations of DNA fragments. However, none of these reports investigated in detail the behaviour of the ITP stage. This paper reports the first known investigation into the behaviour of DNA under conditions of miniaturised isota- chophoresis. As part of the study an electrolyte system was devised which enabled DNA to be isolated and migrated isota- chophoretically on a poly(methyl methacrylate) (PMMA) chip device. The developed system was subsequently tested with sam- ples of salmon sperm DNA and human genomic DNA extracted from whole blood. 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.01.026

Miniaturised isotachophoresis of DNA

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Page 1: Miniaturised isotachophoresis of DNA

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Journal of Chromatography A, 1156 (2007) 154–159

Miniaturised isotachophoresis of DNA

Jeff E. Prest a,∗, Sara J. Baldock a, Philip J.R. Day b, Peter R. Fielden a,Nicholas J. Goddard a, Bernard J. Treves Brown a

a School of Chemical Engineering and Analytical Science, The University of Manchester,P.O. Box 88, Manchester M60 1QD, UK

b CIGMR, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK

Available online 12 January 2007

bstract

This paper presents the findings of a feasibility study investigating the behaviour of DNA under conditions of miniaturised isotachophoresis. Anlectrolyte system comprising a leading electrolyte of 5 mM perchloric acid at pH 6.0 and a terminating electrolyte of 10 mM gallic acid was devisednd used to perform isotachophoresis of DNA containing samples on a miniaturised poly(methyl methacrylate) device. Under such conditions itas found that no separation of DNA fragments was observed with the substance migrating instead as a single isotachophoretic zone. Whilst such

result shows the method is unsuitable for analysis DNA it offers significant potential as a means of sample preparation for subsequent analysissing another method. This is because the single zone of DNA formed is preconcentrated to a constant concentration governed by the leading ionnd is separated from all species with different effective electrophoretic mobilities.

2007 Elsevier B.V. All rights reserved.

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eywords: DNA; Isotachophoresis; Sample preparation; Miniaturisation; Micr

. Introduction

Electrophoretic methods have proven themselves to be use-ul tools in analysing nucleic acids. In particular, the use ofapillary zone electrophoresis (CZE) and capillary gel elec-rophoresis (CGE) have been found to be effective methods forhe separation of DNA fragments [1,2]. These methods have alsoroven readily amenable to implementation on a miniaturisedcale [3,4]. Miniaturisation offers a range of benefits includ-ng improved analytical performance, reduced analysis timesnd the relative ease of performing parallel analyses. These lat-er two features allow for high throughput operations and were

major part of the reason why these electrophoretic methodsere widely used in the project to map the human genome

5].Isotachophoresis (ITP) is another member of the family of

lectroseparation techniques, albeit one that is somewhat less

ommonly encountered than CZE. ITP does however offer aumber of useful features not found in CZE. The most usefulf these is the ability to control separation parameters by vary-

∗ Corresponding author. Tel.: +44 161 306 8900; fax: +44 161 306 4896.E-mail address: [email protected] (J.E. Prest).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.01.026

ce; Chip

ng the electrolyte system used. The concentration of the leadinglectrolyte governs the concentration that all of the sample zonesdopt, enabling ITP to be used as a method to preconcentrateilute samples. Like many other electroseparation methods ITPan be readily miniaturised. When performed in such a formathe technique has been used as both a separation method in itswn right [6,7] or as a sample preparation step prior to perform-ng a CZE separation [8,9]. Miniaturised ITP has been used withwide variety of samples as evidenced by the range of applica-

ions shown in a recent review on the topic [10]. However, to datehere have been few reported uses of miniaturised ITP involvingNA samples. The use of the method has seen some use as a pre-

oncentration method applied either conventionally (ITP–ZE)11] or as transient ITP (TrITP–CGE) [12,13] for separations ofNA fragments. However, none of these reports investigated inetail the behaviour of the ITP stage.

This paper reports the first known investigation into theehaviour of DNA under conditions of miniaturised isota-hophoresis. As part of the study an electrolyte system wasevised which enabled DNA to be isolated and migrated isota-

hophoretically on a poly(methyl methacrylate) (PMMA) chipevice. The developed system was subsequently tested with sam-les of salmon sperm DNA and human genomic DNA extractedrom whole blood.
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atogr. A 1156 (2007) 154–159 155

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Table 1Separation program used to carry out miniaturised isotachophoretic separations

Step Time (s) Current (�A) Valve status

A B C D E

1 20 0 x o x x o2 20 0 x o x o x3 1 0 x x o o x4 0.5 0 o x x o x5 0.5 0 o x x x o6 80 25 x x x x x7 1000 10 x x x x x

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. Experimental

.1. Instrumentation

The miniaturised PMMA separation device used in this workas fabricated using a previously described direct milling pro-

edure [14]. Sealing of the device was achieved by meansf a piece of self-adhesive, 400 �m thick, polyester laminateRitrama, Monza, Italy). The device comprised of two mainhannels, one linking the cross to the junction point, which hadlength of 57 mm, and the second running between the junc-

ion point and well B. This latter channel was 200 �m widend 300 �m deep and incorporated a conductivity detector withn-column 75 �m diameter platinum wire electrodes (Aldrich,illingham, Dorset, UK) arranged in an opposed configuration.he detector was located at a distance of 44 mm from the junc-

ion point. All other channels were 300 �m wide and 300 �meep. A schematic diagram of the channel network is shown inig. 1. The overall size of the device was 78 mm wide by 78 mm

ong.A PS350 high voltage, 5 kV power supply (Stanford Research

ystems, Sunnyvale, CA, USA), configured to supply negativeoltages was used to provide the constant currents required torive the separations. Conductivity detection was achieved usingsystem built in-house, which uses capacitive coupling to iso-

ate the low voltage detection circuitry from the high separationoltages. Electrolyte and sample loading was performed using aravity feed hydrodynamic fluid transport system detailed in anarlier paper [14]. However, a change was made to the sam-le reservoir in this study. To reduce the volume of sampleequired a 1000 �l disposable pipette tip was used for this pur-ose in place of a barrel of a 2.5 ml disposable syringe. Controlf the power supply, fluid transport system and data acquisi-ion from the detector was carried out using a standard PC withrograms written in-house using LabVIEW software (version

.1, National Instruments, Austin, TX, USA). Full details of thenstrumentation have been previously described by the authors15].

ig. 1. Schematic diagram of the channel network in the miniaturised PMMAeparation device. All channels are 300 �m wide and 300 �m deep with thexception of that running from well B to the junction point which is 200 �mide and 300 �m deep. Letters A, B, C, D and E refer to the wells into which

he inlet/outlet connections to the device are made. Sample enters through well, leading electrolyte through B and terminating electrolyte through C. Wellsand E exit to waste.

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ere, x: closed; o: open.

.2. Separation Conditions

A seven step control program, shown in Table 1, was usedo perform all of the separations carried out during this inves-igation. The system is flushed and the device is loaded witheading electrolyte in steps 1 and 2. Terminating electrolyte ishen loaded in step 3. Steps 4 and 5 inject sample into the device.he former step positions the sample at the cross whereas the

atter fills the separation channel between the cross and junc-ion point, thus giving an injection volume of 5.1 �l. The actualsotachophoretic separation begins in step 6 with a current of5 �A applied between wells B (ground) and C. In step 7, thepplied current is reduced to 10 �A, to slow down the separatedones for detection purposes.

.3. Chemicals

The compositions of the electrolytes used in this work areiven Table 2. The leading electrolyte was produced using per-hloric acid (70%, Riedel-de Haen, Gillingham, Dorset, UK)ith Mowiol (40-88, Aldrich, Gillingham, Dorset, UK) added

o suppress electroosmotic flow. The pH of the leading elec-rolyte was adjusted using 2-methylbenzimidazole (98%, Fluka,illingham, Dorset, UK). The terminating electrolyte was gallic

cid monohydrate (>99%, Acros, Loughborough, UK). Modelamples were prepared using low molecular weight salmonperm DNA (Fluka). Samples and electrolytes were prepared

sing >18 M� water (Elga Maxima Ultra Pure, Vivendi Waterystems, High Wycombe, UK).

able 2omposition of the electrolyte system developed to allow the isotachophoreticigration of DNA

lectrolyte Leading Terminatingon Perchlorate Gallateoncentration (mM) 5 10H buffer MBI –H 6.0 –dditive Mowiol –oncentration (g l−1) 0.5 –

BI: 2-methylbenzimidazole.

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1 atogr. A 1156 (2007) 154–159

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Fig. 2. Isotachopherogram produced when a sample containing 125 �g ml−1

salmon sperm DNA was analysed using miniaturised ITP. Leading electrolyte5ie

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56 J.E. Prest et al. / J. Chrom

. Results and discussion

.1. Design of Electrolyte System

Previously there have been no reported studies into theehaviour of DNA under conditions of miniaturised ITP. There-ore, it was necessary to develop a new electrolyte system forse in this study. As it was not known what kind of separa-ion, if any, would occur when such a procedure was attemptedt was thought useful to develop an electrolyte system suitableor use in sample preparation situations. In such circumstances,he primary demand on an electrolyte system is to allow a pureone of the particular species of interest to be produced, ratherhan allowing the simultaneous separation and determinationf a wide range of species. One of the useful features of iso-achophoresis is the ability to tailor the electrolyte system toliminate interference from other species present in the sampleatrix [16]. This can be done by restricting the number of species

n a sample which migrate isotachophoretically by using a lead-ng electrolyte and terminating electrolyte with only a relativelymall mobility difference between them.

The findings of a previous study using free solution ITP aspreconcentration method for a CGE separation indicated thatNA exhibits a mobility a little higher than that of acetate atH 8.3 [17]. Therefore, acetate could be a possible terminatingon. However, it was thought prudent to use a slightly slowerpecies, gallate, in case DNA exhibited a slower than expectedobility under conditions of miniaturised ITP. The leading ion

elected for this study was perchlorate. This species has a lowerobility than the ubiquitous inorganic anions chloride, nitrate

nd sulphate. Thus, these species should not cause any interfer-nce. DNA exhibits a constant charge-to-size ratio between pH.0 and 8.0 [18]. This means that the effective mobility over thisange should be constant and similar to that at pH 8.3 referredo above. For this work, a leading electrolyte pH of 6.0 wassed. This pH was selected to minimise potential interferencerom carbonate which arises from dissolved atmospheric carbonioxide. A low concentration (5 mM) leading ion was decidedpon to hopefully negate any possible precipitation problems.ull details of the electrolyte system can be found in Table 2.

.2. Separations

Prior to performing any experiments in this study it was notnown what the outcome would be when samples of DNA wereubjected to miniaturised ITP. This was because it had been pre-iously suggested that the use of capillary scale ITP was nothe ideal method for the separation of oligonucleotides (andy inference DNA) [19]. However, gel based ITP had previ-usly been shown to offer some potential for the separation ofNA fragments [20]. Therefore, initially it was necessary toerform a series of experiments to identify what kind of sep-ration arose. These preliminary experiments were carried out

sing low molecular weight salmon sperm DNA (<2000 bp) toroduce samples. Using the devised electrolyte system it provedossible to get the DNA migrating isotachophoretically betweenhe leading and terminating electrolytes. An example of such a

ce

i

mM HClO4, 0.5 g l−1 Mowiol, pH 6.0 (2-methylbenzimidazole). Terminat-ng electrolyte 10 mM gallic acid. LE: leading electrolyte; TE: terminatinglectrolyte.

esult, for a sample containing 125 �g ml−1 salmon sperm DNA,s shown in Fig. 2.

From the illustrated result it can be see that the DNA pro-uced a sharp zone when subjected to miniaturised ITP. Thisesult is somewhat different to that observed in a previous reportf using capillary scale ITP to analyse nucleic acids, includingNA [21]. In this previous study, the aim was to separate DNA

ragments. To try and achieve this an ampholyte solution wasdded to the samples and this substance contributed to complex-ty of the observed results recorded using a potential gradientetector. This previous study used a leading electrolyte at pH.9 and also suffered some problems with carbonate interfer-nce. As mentioned above such interference was not a problemn the current work due to the electrolyte system developed. Theesults observed in the current study suggested that the DNAas migrating with a relatively homogenous effective mobility

nd that there was essentially no separation of the DNA intoragments. This result indicates the method was unlikely to bef use for separating DNA fragments. However, from a samplereparation perspective it was a very promising result as it indi-ated that all of the DNA was being concentrated into a singleone which could then be used in another procedure. Such anperation may be another separation process such as CZE orn amplification process such as a polymerase chain reactionPCR). As ITP is a separation technique this zone of DNA wille separated from any other substances present in samples whichave different effective mobilities. Such a feature is of signifi-ant importance if the DNA is subsequently being subjected toCR. This is because the amplification can suffer a loss of effi-

iency by the presence of certain species such as heme, lipids ornzymes [22,23].

The isotachophoretic migration of DNA was found to occurn a reproducible manner. Results observed with samples of

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almon sperm DNA showed a good level of repeatability. Whenen consecutive runs were carried out using a sample con-aining 125 �g ml−1 DNA the observed relative step heightRSH) ± standard deviation (SD) was found to be 0.673 ± 0.011.n this work, the RSH was taken as the ratio of the sample stepeight to the height of the terminating step. When conductiv-ty detection is used in ITP the step height provides qualitativenformation about the substances being analysed. Good repro-ucibility was also witnessed in the quantitative informationontained in the isotachopherograms, the zone lengths. The tenepeat runs of the 125 �g ml−1 salmon sperm DNA producedones ± SD with a length of 19.8 ± 0.42 s. The use of a lowoncentration, 5 mM, leading electrolyte allows for relativelyast analysis times. For example, the result shown in Fig. 2 waschieved in just over 3 min. This separation time included the2 s to complete the injection program, so that the actual isota-hophoretic separation was completed in under 160 s. Thus, itan be seen the miniaturised ITP is eminently suitable for uses a high throughput sample preparation method.

To investigate the range of DNA concentrations over whichhe method could be used a series of nine samples were analysed.our replicate runs were performed with each of the samples,hich contained salmon sperm DNA concentrations ranging

rom 9.5 to 750 �g ml−1. The results obtained from these experi-ents were used to check the linearity of the method by means ofcalibration curve. It was found that good linearity was obtainedith a correlation coefficient of 0.998 calculated. The parametersf the curve, produced using weighted linear regression, werehat the slope was 0.132 ± 0.001 s ml �g−1 and the intercept was.94 ± 0.37 s. The errors shown for both of these parameters rep-esent the SDs. Using the regression equation a limit of detectionLOD) for salmon sperm DNA was calculated to be 8.4 �g ml−1.his value was calculated using the intercept, which representsn estimation of the blank, plus three times the standard devia-ion associated with this parameter. The calculated LOD givesn indication of the concentration of DNA which is necessaryo be present in a sample that is being pretreated so that it cane detected reliably using the conductivity detector used in thisork. This figure is not necessarily the minimum concentration

equired to produce an isotachophoretic zone. Using an alterna-ive detection method such as UV-absorbance may allow shorterones to be detected. Such an effect can occur because UV-bsorbance is a specific detection method and conductivity a

niversal detection method [24]. To realise the potential ben-fits from such an approach would require an amendment ofhe electrolyte system used in this work as it would be neces-ary to change from using gallate to using a non-UV absorbing

aIad

able 3esults obtained with human genomic DNA samples

ample Relative step height ± SD

n-house 1 0.629 ± 0.039n-house 2 0.686 ± 0.076n-house 3 0.675 ± 0.020epnel 1 0.632 ± 0.019

a Samples made up to 1000 �l with deionised water prior to analysing with ITP sta

. A 1156 (2007) 154–159 157

pecies as the terminating ion. The use of a specific detectoruch as UV-absorbance or fluorescence (which offers the possi-ility of higher sensitivity [25]) may be useful if using ITP assample pretreatment method. This is because the species of

nterest can be bracketed by substances which are not detectedy a specific detector. Whilst bracketing has been used in theast to improve quantification [26], the process can also besed to offer an accurate means of determining when to makehe cut in the isotachophoretic stack to remove the requiredpecies.

One of the useful features of ITP which makes it suitable forample preparation purposes is the fact that the concentrationf the zones in isotachophoretic stack are of a fixed concen-ration. This concentration is governed by the choice of thelectrolyte system and is approximately that of the leading ion.his feature means that for example the isolated DNA can be

ransferred for a subsequent operation such as PCR amplificationn a volume added basis. This simplifies the transfer operations it eliminates the need to determine the concentration of theNA. However, this same feature can make quantification ofNA using ITP somewhat problematic. As the zones formed

re of a fixed concentration, quantification can be achieved whensing conductivity detection by measuring the length of the zone.he fixed concentration of the zones means the higher the con-entration present in the sample solution the longer the zoneength produced. The problem with DNA samples is the poly-

eric nature of the substance. Thus, different samples can haveariable molecular masses. Thus, although a calibration curveas produced for the salmon sperm DNA this calibration willot necessarily hold for other DNA samples. For example, thealmon sperm DNA used in this work has a mass of approx-mately 1.3 × 106 Da [27] whereas genomic human placentaNA (Sigma, Gillingham, Dorset, UK) has a mass of approxi-ately 10 × 106 Da. If DNA samples from a different source are

nalysed different zone lengths will be obtained for equivalentample concentrations.

This type of problem arises when any type of polymeric mate-ial is analysed using ITP. However, it was overcome to an extenthen using ITP to analyse carboxymethylcellulose polymersy introducing an ‘equivalents of carboxyl group’ factor intohe calibration [28]. To implement this approach did, however,ecessitate a prior analysis of the samples using a potentiometricitration. An alternative approach used for the isotachophoretic

nalysis of polyphosphates was to use an internal standard [29].f quantification of DNA was required it may be possible topply one of these approaches. Again the use of an alternativeetection method may be beneficial in reducing this problem.

Step length ± SD (s) Sample volumea (�l)

8.4 ± 0.2 5010.4 ± 0.6 5012.5 ± 0.9 60

8.1 ± 0.3 45

ndard deviations based on four replicates.

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58 J.E. Prest et al. / J. Chrom

To further investigate the behaviour of DNA when subjectedo miniaturised ITP a series of analyses were made using a num-er of samples of genomic human DNA. The samples used wereroduced from whole human blood. These samples were puri-ed by either an in-house method involving a phenol/chloroformxtraction or using a commercial kit (Tepnel Life Sciences,anchester, UK). Table 3 lists the RSHs and zone lengths

bserved with the genomic DNA samples. The volumes of theamples analysed are also given in this table. These values areifferent for the different samples as different amounts of thendividual samples were available and the maximum possibleolume was analysed. Fig. 3 shows an example of an isotacho-herogram obtained with one of these samples (in-house 2).enerally, clear results exhibiting only a single obvious stepere obtained with the genomic samples. However, with in-ouse sample 1 an additional short zone with a RSH ± SD of.331 ± 0.019 was noted. Longer analysis times were observedith all of the genomic samples compared to those of the salmon

perm samples. For example, the rear of the leading zone in theesult shown in Fig. 3 with in-house sample 2 was detected after27 s whereas in the example in Fig. 2 with the salmon spermNA the same feature was detected after 178 s. This finding

ndicates the presence of high mobility species in the genomicamples. Such species are likely to be inorganic salts such ashloride, nitrate or sulphate. These species all have higher effec-ive mobilities than the perchlorate leading electrolyte. Thus,hey will not produce an isotachophoretic step but will instead

igrate within the leading electrolyte, resulting in a lengtheningf this zone.

As in ITP qualitative information is contained in the stepeights produced, if what is detected with the genomic samples

s DNA, there should be agreement between the RSHs observedith these samples and that of the salmon sperm DNA. To checkhether this was the case the results were subjected to a Stu-

ig. 3. Analysis of a sample of genomic human DNA from whole blood analysedsing miniaturised ITP. Conditions used as given in Fig. 2.

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. A 1156 (2007) 154–159

ent’s t-test. This statistical test was used to compare the resultsielded by each of the genomic samples to those obtained withalmon sperm samples. The value used as the RSH ± SD for thealmon sperm DNA in these tests was taken as 0.652 ± 0.023n = 36), which represents the results observed in producing thealibration curve. The results of the tests for all four genomicamples yielded t-values under the appropriate critical t-valuest the 95% level. Thus, it could be said that statistically the stepsbserved were the same as with the salmon sperm samples andhat what was being seen with the genomic samples was notignificantly different from salmon sperm DNA.

Due to the earlier mentioned problems regarding calibra-ions, meaningful concentrations of the genomic samples couldot be calculated. The differences in sample volumes analysedeant a simple comparison of zone lengths could not be used

o determine the concentrations of DNA present in the sam-les compared to one another. However, it was thought that thealibration curve produced for the salmon sperm DNA coulde used to enable a comparison of the concentrations of DNAresent in the original genomic DNA samples to be made. Whenhis was done it was found that in-house sample 3 had theighest concentration of DNA followed by in-house sample 2nd in-house sample 1 had the lowest concentration of DNA.his result was in agreement with the concentrations of theamples determined using a ND-1000 UV spectrophotometerNanoDrop Technologies, Wilmington, DE, USA), which werehat in-house sample 1 contained 113 �g ml−1 of DNA, sam-le 2 contained 202 �g ml−1 of DNA and sample 3 contained34 �g ml−1 of DNA. These results indicate that the DNA fromhe genomic samples was successfully separated from the sam-les and concentrated up into isotachophoretic zones. The DNAoncentration of the sample extracted using a commercial kitas found to be 350 �g ml−1 using the NanoDrop instrument.owever, the results obtained using miniaturised ITP suggested

he concentration was between those of in-house samples 1 and. It is not known whether this discrepancy was related to thextraction method used or due to the DNA in this sample havingdifferent mass.

. Conclusions

When DNA samples were subjected to miniaturised ITP sep-rations they were found to form an essentially homogenousone with little evidence of fragmentation. Whilst such a results of little use from the point of view of analysing the DNA itffers potential as a sample preparation method. This is becausehe zone of DNA formed has a fixed concentration governedy the electrolyte system. This means that the DNA zone cane transferred to a subsequent further operation on a volumeasis and also allows for preconcentration of dilute samples. Thisdvantage is seen as being particularly beneficial for preparingamples for PCR amplification. The separation mechanism ofTP allows for the elimination of unwanted matrix components

o be achieved. In the current study, such an effect was achievedith genomic DNA samples extracted from whole human bloodut it should be possible to use the method to for example isolateNA from cell lysis products.
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The use of miniaturisation technology allows for the proce-ure to be carried out rapidly. With the device used in this workamples produced using salmon sperm DNA were separated innder 200 s whereas those containing genomic DNA were sep-rated in under 280 s. Thus, the method has potential for highhroughput sample preparation. Indeed, if required these figuresould be improved upon further with a different design of device.he times indicated also include the preparation steps of loading

he device. This part of the process can be achieved rapidly as theeparations are performed in free solutions rather than gels. Therocedure is also easy to implement requiring relatively simplenstrumentation.

cknowledgement

This work was funded by the Medical Research CouncilUK).

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