Digital Polymerase Chain Reaction Measured pUC19 Marker as

Dec 6, 2012 - A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system fo...
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Digital Polymerase Chain Reaction Measured pUC19 Marker as Calibrant for HPLC Measurement of DNA Quantity Daniel G. Burke,*,† Lianhua Dong,‡ Somanath Bhat,† Michael Forbes-Smith,† Shuang Fu,† Leonardo Pinheiro,† Wang Jing,‡ and Kerry R. Emslie† †

National Measurement Institute, Lindfield, Australia 2070 National Institute of Metrology, Beijing, China, 100013

‡

S Supporting Information *

ABSTRACT: Digital polymerase chain reaction (dPCR) is potentially a primary method for quantifying target DNA regions in a background of nontarget material and is independent of external calibrators. Accurate dPCR measurements require singlemolecule detection by conventional PCR assays that may be subject to bias due to inhibition, interference, or sequence-derived PCR inefficiency. Elimination or control of such biases is essential for validation of PCR assays, but this may require a substantial investment in resources. Here we present a mechanism for DNA quantification that does not require PCR assay validation in situations where target DNA quantity is high enough to be measured by physical techniques such as quantitative highperformance liquid chromatography (HPLC) or electrophoresis. A commercially available DNA marker derived from pUC19 was quantified by dPCR and was then used to calibrate an HPLC measuring system for quantifying a DNA amplicon that had a high content of guanidine and cytidine. The dPCR-calibrated HPLC measurement was verified by independent measurement using isotope dilution mass spectrometry (IDMS). HPLC quantification, calibrated with dPCR or IDMS measured DNA markers, provides an effective method for certifying the quantity of genetic reference materials that may be difficult to analyze by PCR. These secondary reference materials may then be used to validate and calibrate quantitative PCR measurements and thus could expand the breadth of applications for which traceability to the International System of Units is possible.

D

would facilitate quantity measurements of genetic reference materials that are globally comparable. DNA ladders are commonly used as reference markers for determining the size of double-stranded DNA fragments using techniques such as gel electrophoresis. The DNA fragments in a ladder typically range in size from less than one hundred base pairs up to a few thousand base pairs, and these fragments are ideally suited for use as calibrants for concentration measurements in HPLC and CE systems. One such DNA marker readily available in purified form is the MspI digest of pUC19. Since this plasmid has a GC content of 52% the potential PCR biases associated with GC-rich regions can be avoided and accurate quantity measurements may be made using PCR-based systems. The accurately quantified DNA solution may then be used to calibrate measuring systems such as HPLC or CE that are less affected by GC content. The purpose of this study was to quantify DNA fragments in the MspI digest of the plasmid pUC19 by dPCR and to use these fragments as calibrants to quantify GC-rich templates by

igital polymerase chain reaction (dPCR) measurements of target DNA use limiting dilution and the ability to detect single molecules to measure the quantity of DNA without reference to a standard.1,2 Measurement of DNA quantity of genetic reference materials using dPCR thus has the potential to standardize genetic measurements to the mole in the International System of Units (SI) making all such measurements internationally comparable.3 However, PCRbased measurements can be affected by suboptimal amplification efficiency, especially of guanidine and cytidine (GC)-rich regions,4−8 or due to presence of PCR inhibitors.9,10 Indeed, it has been shown that clinical diagnostic measurements using methylated and GC-rich templates may be incorrect due to temperature-dependent effects in PCR.8 Though several mechanisms to overcome these problems have been proposed, for example, use of GC enhancers, amplification of some DNA sequences with high GC content remains challenging.11 High-performance liquid chromatography (HPLC), fluorescent labeling, gel electrophoresis, and capillary electrophoresis (CE) can all provide alternative means of measuring DNA quantity.12,13 However, these techniques require a calibrant and have widely varying accuracy. Development of a suitable calibrant with defined mass concentration traceable to the SI © 2012 American Chemical Society

Received: October 9, 2012 Accepted: December 6, 2012 Published: December 6, 2012 1657

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Figure 1. Linear map of the HindIII digest of pUC19 showing the relative positions of qPCR target sequences.

During this work, we found that a number of commercial PCR master mixes were contaminated with interfering DNA that caused a variable measurement bias when present. Though this has been observed previously, none of the common PCR master mix manufacturers discloses whether their reagents are tested for the presence of DNA that may interfere with the quantitative measurement for which they are designed.18

HPLC. The GC-rich template chosen was a 304 bp amplicon with a GC content of 73% from a region of the human TLX3 gene that may be diagnostic for pre-B cell acute lymphoblastic leukemia.14 Potential biases in HPLC measuring systems are caused by interference from unresolved DNA fragments, differences in matrix effects between sample and calibration solutions, and differences in detector response factors between analyte and calibrant. Since HPLC resolution of DNA molecules is based primarily on the number of phosphate groups and UV detector response is determined by absorption coefficient, DNA sequence variables such as GC content may have only a minor contribution to biases in this measuring system.15 Thus, high GC content that stabilizes doublestranded DNA is an advantage for HPLC but may result in incomplete denaturation and significant bias in PCR-based measurements. To validate the HPLC measuring system incorporating the dPCR measured calibrant an independent measurement of DNA quantity was needed. Isotope dilution mass spectrometry (IDMS) has recently been shown to be an accurate method for quantifying DNA materials, so the HPLC measurement was verified using IDMS as biases in this technique are not correlated with those of dPCR or HPLC measurement of double-stranded (ds) DNA fragments.16 The IDMS measuring system consists of enzymatic hydrolysis to deoxynucleotide monophosphates (dNMPs) followed by addition of stable isotope labeled dNMP internal standards and measurement of the peak area ratios of fragment ions obtained from the molecular ions of the labeled and unlabeled analytes. The DNA quantity is calculated using a calibration curve or the exact matching technique where in both cases the observed peak area ratios are calibrated by dNMP reference materials.17 Biases in the IDMS measuring system may be caused by incomplete enzymatic digestion of the DNA strands, discrimination between natural and labeled dNMPs during sample processing, differences in matrix effects between sample and calibration solutions, and by interference on liquid chromatography−mass spectrometry (LC−MS). These biases are completely different from those encountered in dPCR measurement of DNA quantity; here, the major bias originates from measuring partition volume (provided that a sufficient number of reactions is assessed), and there are also potential biases from denaturation during sample loading, nonspecific amplification, or incomplete denaturation during PCR, all of which are affected by DNA sequence characteristics. Ideally dPCR can measure the quantity of a specific target region in a background of nontarget DNA, whereas IDMS measures the entire quantity of DNA in the test solution.

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EXPERIMENTAL SECTION pUC 19 DNA Marker. The DNA marker used for this work was plasmid pUC19 digested with MspI (Fermentas marker 23) supplied in 100 μL portions at a nominal concentration of 500 ng/μL in 10 mM Tris−HCl pH 7.6 containing 1 mM EDTA; it is recommended for sizing and approximate quantification of small linear double-stranded DNA fragments. The plasmid sequence used was Genbank accession number L09137.2; it has 2686 base pairs (bp) containing 13 MspI restriction sites yielding 13 fragments with sizes in bp of 501, 489, 404, 331, 242, 190, 147, 111, 110, 67, 34, 34, and 26. These fragment sizes cover the range expected of amplicons that may be synthesized for quantitative measurement of gene exons since 80% of exons are less than 200 bp in length.19 Ten portions of the 500 ng/μL pUC19 MspI digest mixture were pooled then gravimetrically diluted 10-fold to give 10 mL with a nominal total concentration of 50 ng/μL; 100 μL portions of the diluted DNA were dispensed into 100 vials and stored at −20 °C. The predicted restriction fragment coordinates and their nominal concentrations in the 50 ng/ μL vials, calculated from the molecular weight fraction of the fragment, are presented in Supporting Information Table S-1 (note the prefix “S” before a table or figure number denotes its presence in the Supporting Information). Digital PCR. Primer pairs and probes were designed using Beacon Designer 7.5 to amplify a 133 bp (assay 1) and an 87 bp (assay 2) segment within the 501 bp and 242 bp fragments of the pUC19/MspI digest, respectively. The 501 bp and 242 bp fragments of the pUC19/MspI digest were specifically targeted since they cover the size range of typical amplicon reference materials prepared in our laboratory. Both of these fragments fall within the ampicillin resistance gene which is a common component of engineered bacterial vectors. Primer pair and probe sequences are given in Supporting Information Table S-2, and positions of target sequences are shown in Figure 1. Digital PCR analysis was performed using both assays in simplex on the BioMark system (Fluidigm, San Francisco) with the 12.765 digital arrays, i.e., each array had 12 panels of 765 partitions. Eight of the pUC19/MspI digest 50 ng/μL vials (vial 1658

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Quantitative HPLC. The 304 bp amplicon with a GC content of 73% from a region of the human TLX3 gene that may be diagnostic for pre-B cell acute lymphoblastic leukemia was prepared by end-point PCR (primers given in the Supporting Information), precipitated from ethanol, and dissolved in TE0.1 buffer.14 The purified material (20 μL, 160 ng/μL) was fractionated by ion-exchange HPLC and was ultrafiltered (Pall Nanosep 3K omega). The retentate was dissolved in 200 μL of TE0.1 buffer and analyzed by quantitative HPLC and by IDMS using the exact matching technique.17 Quantitative HPLC measurements were made using a Dionex Ultimate 3000 RSLC nano system with a Dionex PepSwift monolithic PS−DVB column 250 mm long × 0.2 mm internal diameter at 60 °C, a flow rate of 2.75 μL/min, and UV detector monitoring 260 nm. The initial mobile phase composition was 50% of A (200 mM triethylammonium acetate) and 50% of B (20% acetonitrile in 200 mM triethylammonium acetate); the gradient was linear to 65% B in 3 min, then to 85% B at 12 min, 95% B at 13 min, isocratic for 7 min, and re-equilibrated to 50% B for 10 min. The TLX3 304 bp amplicon was analyzed in three sets of five replicate measurements with each set bracketed with five replicate measurements of the pUC19 reference material. The concentration was calculated using eq 1:

numbers 8, 16, 24, 40, 48, 64, 72, and 96) were selected for homogeneity analysis. A 10 μL portion from each vial was gravimetrically diluted to give a nominal 677 copies/μL, and a separate subsample for assay 1 and assay 2 was taken for dPCR measurement. Each dPCR measurement was replicated five times to give a total of 40 dPCR measurement for each assay and a total of 80 measurements overall. The 80 measurements were spread over eight 12.765 chips with each chip having five panels of assay 1, five panels of assay 2, one panel of negative control for assay 1, and one panel of negative control for assay 2. For each chip, the reaction mixture for each assay was prepared in a total volume of 70 μL consisting of 35 μL of TaqMan Fast Universal PCR master mix (2×), No AmpErase UNG part no. 4352046, 3.5 μL of DA loading reagent (Fluidigm), 28 μL of diluted pUC19/MspI, required volumes of primers and probes to give optimized concentrations as specified in Supporting Information Table S-2, and TE0.1 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to give a total volume of 70 μL. To minimize the uncertainty from pipetting, the pUC19/MspI template was added gravimetrically to a premix containing all other components. The final reaction mix (10 μL) was transferred into each sample inlet on the digital array with approximately 4.6 μL of the reaction mix distributed throughout the partitions within each panel using an automated NanoFlex IFC controller (Fluidigm, South San Francisco). Thermocycling parameters were 2 min of activation at 95 °C followed by 45 cycles of 10 s at 95 °C and 30 s at 60 °C. The copy number measurement was calculated from the number of positive partitions of the possible 765 modeled on a Poisson distribution to account for multiple copies per well in a total volume of 4.6 μL (765 partitions each 6 nL). The standard uncertainty for digital PCR measurements was calculated as published previously;1 expanded uncertainty was obtained by multiplying the standard uncertainty by a coverage factor calculated using the Welch−Satterthwaite equation to give a 95% level of confidence.20 For the 6 month stability measurement a different master mix was used as DNA contamination was found in master mix from the original supplier by treating it with DNase I. The DNase Itreated master mix was not used since complete inactivation of DNase I is required for dPCR measurements and this was difficult to control. For these dPCR measurements, three independent gravimetric dilutions were prepared from a single vial of 50 ng/μL pUC19/MspI digest that had been stored at −20 °C and a separate subsample of the diluted material was taken for assay 1 and assay 2. Each dPCR measurement was replicated five times to give a total of 15 dPCR measurement for each assay and a total of 30 measurements overall. The 30 measurements were spread over three 12.765 chips with each chip having five panels of assay 1, five panels of assay 2, one panel of negative control for assay 1, and one panel of negative control for assay 2. For each chip, the reaction mixture was prepared in a total volume of 80 μL consisting of 40 μL of Stratagene brilliant II QPCR master mix cat. 600804, 4 μL of DA loading reagent (Fluidigm), 32 μL of diluted pUC19/MspI, required volumes of primers and probes to give optimized concentrations as specified in Supporting Information Table S2, and TE0.1 buffer to give a total volume of 80 μL. Preparation of PCR reaction mix was the same as for homogeneity measurements. Thermocycling parameters were 10 min of activation at 95 °C followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C.

Csamp =

A samp IVstd Cstd A std IVsamp

(1)

where Csamp is the concentration of the target fragment in the sample solution (ng/μL), Asamp is the peak area of the target fragment in the sample, IVsamp is the injection volume of the sample solution (μL), Cstd is the concentration of the matching fragment in the standard solution (ng/μL), Astd is the peak area of the target fragment in the standard, and IVstd is the injection volume of the standard solution (μL). The uncertainty of the concentration measurement was calculated using eq 2. The injection volumes were identical for sample and standard, so any volume error would cancel thus allowing this factor to be excluded from the uncertainty calculation. uCsamp = Csamp

⎛ u A ⎞2 ⎛ u A ⎞2 ⎛ uC ⎞2 ⎜⎜ samp ⎟⎟ + ⎜ std ⎟ + ⎜ std ⎟ ⎝ A std ⎠ ⎝ Cstd ⎠ ⎝ A samp ⎠

(2)

uAsamp/Asamp is the relative standard deviation of the sample peak area, uAstd/Astd is the relative standard deviation of the standard peak area, and uCstd/Cstd is the relative standard uncertainty of the pUC19 concentration from digital PCR. Ion-Exchange HPLC. Ion-exchange HPLC was performed using a Shimadzu Prominence system with a Waters Gen-Pak FAX 4.6 mm × 100 mm column at 55 °C and flow rate 0.75 mL/min. The mobile phases were A, 25 mM Tris−HCl, 1 mM EDTA, pH 8.0, and B, 25 mM Tris−HCl, 1 mM EDTA, 1 M NH4Cl, pH 8.0. The initial mobile phase was 45% A plus 55% B, then a linear gradient to 70% B at 10 min, to 75% B at 15 min, and isocratic to 19 min. The injection volume was 0.1− 100 μL. The detector was a diode array recording the wavelength range of 190−500 nm and cell temperature of 50 °C. Isotope Dilution Mass Spectrometry. Sample blends were prepared by mixing 0.1 g of the ion-exchange HPLC fraction (∼5 ng/μL) and 0.1 g of the stable isotope labeled 1659

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for qualitative investigations. Details of qPCR conditions are given in the Supporting Information.

deoxynucleotide monophosphate (dNMP) mixture; calibration blends were prepared by mixing 0.1 g of a mixed dNMP standard solution with 0.1 g of the stable isotope labeled dNMP mixture. Both sample and calibration blends were digested with a mixture of DNase I and phosphodiesterase for 60 min at 37 °C as previously published.21 The hydrolyzed mixture was ultrafiltered (Pall Nanosep 3K omega), and the filtrate was analyzed on an Agilent 6510 QTOF LC−MS using conditions given in the Supporting Information. Individual dNMP concentrations in the mixed dNMP standard solution were prepared to match the dNMP concentrations obtained from complete hydrolysis of the ∼10 ng/mg 304 bp amplicon and are given in the Supporting Information. Equation 3 was used to calculate the mass fraction of each nucleotide from replicate analyses of each sample blend: wX = wZ

mY mZ RB mX mY ,c RB,c

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RESULTS AND DISCUSSION Measurement of pUC19 Concentration by dPCR. Results for the eight 50 ng/μL pUC19/MspI digest vials selected for dPCR quantification to evaluate vial homogeneity are presented in Table 1. The average concentrations measured Table 1. dPCR Measured Concentrations (Average of Five) of pUC19 Fragments 501 bp and 242 bp in Eight Vials assay vial 8 24 48 64 16 40 72 96 mean RSD (%)

(3)

where wX is the mass fraction of target molecule in the test solution added to the sample blend (ng/mg), wZ is the mass fraction of reference substance in the standard solution added to the calibration blend (ng/mg), mY is the mass of internal standard solution added to the sample blend (g), mZ is the mass of reference substance solution added to the calibration blend (g), mX is the mass of test solution added to the sample blend (g), mY,c is the mass of internal standard solution added to the calibration blend (g), RB,c is the peak area ratio of selected ions in the calibration blend, and RB is the peak area ratio of selected ions in the sample blend. The mass fraction of the target DNA amplicon was calculated by converting the mass fraction of each dNMP according to its mole fraction in the amplicon using eq 4: wX ,DNA =

2 (cp/μL) 1.95 × 1010 2.07 × 1010 1.85 × 1010 1.99 × 1010 1.49 × 1010 1.71 × 1010 1.87 × 1010 1.88 × 1010 1.85 × 1010 9.8

for the two fragments were not significantly different (t statistic assuming equal variances, α = 0.05), thus indicating that these values could be applied to the entire plasmid and its fragments. The stability of the pUC19/MspI digest at 50 ng/μL was evaluated at 6 months from preparation by measuring five replicates of three independent gravimetric dilutions from a single vial that had been stored at −20 °C. The pUC19/MspI DNA concentration measurements by dPCR at initial time and after 6 months agreed within their expanded uncertainties (Table 2) indicating that the pUC19/MspI digest solutions were stable. At the 6 month measurement, however, a master mix from an alternative supplier was used as contamination was found in a fresh batch of master mix from the original supplier. Subsequent qPCR investigations showed that the contamination could be removed by DNase I pretreatment, but careful optimization of pretreatment conditions was required to avoid interference with PCR measurements. HPLC Quantitation of dsDNA Is Independent of Sequence Size. Quantitation of DNA by HPLC using the external standardization technique requires calibration of the measuring system with a reference material that mimics the compound to be measured. For small-molecule analysis, calibration solutions must be prepared using the synthetic material that has preferably been certified for purity, a certified reference material. In many cases the analyte may be obtained with purity that is fit for purpose from a number of chemical suppliers or from certified reference material suppliers. This is not possible for DNA analysis, as there are an unlimited number of possible DNA sequences under investigation and it is not commercially feasible to prepare and characterize the large variety of DNA molecules that would be required to calibrate each possible target DNA sequence. However, without an appropriate reference material accurate quantification of target DNA and measurement validation is not possible. This seemingly insurmountable conundrum may be overcome by using DNA amplicons that have been quantified by HPLC, using a calibrant with a certified quantity value that is traceable

wX ,dNMP MR,DNA MR,dNMP NdNMP

1 (cp/μL) 1.86 × 1010 2.05 × 1010 1.64 × 10100 1.80 × 1010 1.50 × 1010 1.60 × 1010 1.81 × 1010 1.85 × 1010 1.76 × 1010 9.9

(4)

where wX,dNMP is the mass fraction of dNMP in the hydrolysate (ng/mg), wX,DNA is the mass fraction of double-stranded DNA (ng/mg), MR,DNA is the molecular weight of the TLX3 304 bp amplicon (187 784 g/mol), NdNMP is the number of molecules of the specific nucleotide in the TLX3 304 bp amplicon, and MR,dNMP is the average molecular weight (hydrated) of the specific dNMP (g/mol). Capillary Electrophoresis. Post PCR, the product size (bp) was verified on a 2100 Bioanalyzer (Agilent Technologies, Australia) using a DNA 1000 kit. Samples were prepared as per manufacturer’s instructions.22 Pretreatment of the PCR Master Mix with DNase I. To investigate the positive amplification observed in negative controls, PCR master mixes from four suppliers (Agilent, Roche, ABI, Qiagen, not in order) were pretreated with DNase I prior to combining with the other components of the PCR assay to eliminate any background DNA. Deoxyribonuclease I (DNase I) (Sigma-Aldrich, Australia) was diluted to a final working stock concentration of 2 U/μL in TE0.1 buffer. The DNase I-treated master mix was prepared by adding 1.25 μL of DNase I working stock solution to 100 μL of PCR master mix (final concentration of DNase I was 0.025 U/μL). The contents were mixed well, incubated at 37 °C for 30 min, and then the enzyme was inactivated at 75 °C for 10 min. Ten microliters of DNase I-treated master mix was used in each PCR assay, and the slight dilution factor incurred was not taken into account 1660

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Table 2. Concentration Measurements for pUC19/MspI Candidate Reference Material Using Assays 1 and 2 at Initial Time and 6 Months Later initial assay measd pUC19/MspI concn (copies/μL) expanded uncertainty (copies/μL) coverage factor (k) relative expanded uncertainty (%)

1 1.76 × 1010 0.19 × 1010 2.1 11

6 months 2 1.85 × 1010 0.21 × 1010 2.1 11

1 2.01 × 1010 0.25 × 1010 2.2 12

2 2.07 × 1010 0.24 × 1010 2.1 12

since in this case the detector is based on absorbance at 260 nm integrated over the time of elution from the HPLC column, then molar response factors will be dependent on the fragment/amplicon molar absorption coefficient, and this is determined by base composition and size. Since it is unlikely that both base composition and size of the analyte will match any of the pUC19 MspI fragments, this could invalidate the measurement process. However, the use of mass concentration rather than molar concentration for the calibrant overcomes this problem and is commonly used when measuring DNA concentration by UV absorbance. A rearrangement of the common expression of the Beer−Lambert Law is given in eq 5 where the molar concentration term was converted to mass concentration (γ, g/L) by multiplying by molecular weight (MR, g/mol) and assuming a path length (l) of 1 cm.24

to the SI, as a secondary reference material provided that the uncertainty of the final concentration measurement is fit for purpose. The secondary reference material may also be used to assess recovery from the extraction step where a blank test material is available or by standard addition techniques if not.23 DNA markers are available from a number of suppliers for sizing of predominantly PCR amplicons and may also be used for approximate quantification. The quantity of DNA in these materials is generally obtained by measuring the absorbance at 260 nm and applying the general conversion factor for doublestranded DNA of 50 μg/OD unit; in most cases the accuracy of the quantity is not specified by the supplier. Digital PCR can be used to accurately measure DNA concentration without reference to an external standard provided the assay can repeatably detect single molecules in a known reaction volume. In this case the accuracy of the measurement is determined mainly by the reaction volume given a repeatable PCR assay. When the DNA marker is used for HPLC calibration, resolution of the mixture components and identification of their retention time are necessary in order to assign the correct concentration value for each component. Identification of the pUC19 MspI fragment retention times was accomplished by ion-exchange HPLC fractionation of the digest followed by Sanger sequencing of the individual fragments then analyzing the identity confirmed fragments by capillary HPLC using the same mobile phase gradient as for the mixture. Figure 2 gives

⎡M ⎤ γ = ⎢ R ⎥A ⎣ ε ⎦

(5)

Table 3 shows that MR/ε calculated for each of the pUC19 MspI fragments had a relative standard deviation (RSD) of only Table 3. Calculation of MR/ε for the pUC19 MspI Fragments from the Estimated Molar Absorption Coefficient (ε) and the Molecular Weight (MR)

Figure 2. HPLC chromatogram of 50 ng/μL pUC19 MspI digest using conditions specified in the Experimental Section. The size in base pairs of each fragment is shown above the peak.

length (bp)

ε × 10−3

MR

GC (%)

MR/(ε × 10−3)

501 489 404 331 242 190 147 111 110 67 34 34 26

7870 7730 6350 5250 3820 3000 2340 1760 1740 1060 542 535 417

309569 302217 249639 204585 149563 117436 90882 68622 68004 41437 21047 21044 16101 average RSD (%)

44 56 44 58 49 50 61 50 50 54 59 50 50 52 10.5

39.3 39.1 39.3 39.0 39.2 39.1 38.8 39.0 39.1 39.1 38.8 39.3 38.6 39.1 0.5

0.5% despite a 10.5% RSD in sequence GC content. The molar absorption coefficient (ε) was calculated according to Tataurov et al. and includes estimation of hypochromicity.25 If hypochromicity and other effects were different between analyte and calibrant a bias may be introduced that would be similar in magnitude to the difference in MR/ε. The similarity of the MR/ε value between pUC19 fragments means that only a relatively small difference in calculated analyte concentration would be expected in using any of the fragments as calibrant, but a small bias would be expected if the value of MR/ε of the calibrant was different from that of the

an HPLC chromatogram of the pUC19 MspI digest illustrating that chromatographic resolution was adequate for quantification over the size range. The order of elution of restriction fragments did not correspond to size in contrast to previous observations with ion-pair reversed-phase chromatography of double-stranded DNA.15 The requirements for quantification by external standards are that response factors for analyte and calibrant are the same, and 1661

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Table 4. TLX3 304 bp Amplicon Quantification Using Different pUC19 Fragments as Calibranta pUC19 fragment peak area RSD (%) fragment concn ng/μL TLX3 304 bp ng/μL standard uncertainty ng/μL a

147 14154 3.2 3.1 9.8 0.7

190 17914 3.1 4.0 10.0 0.7

242 22702 3.3 5.1 10.0 0.7

404 34906 3.5 8.5 10.9 0.8

331 31238 3.2 6.9 10.0 0.7

501 44999 3.0 10.5 10.5 0.7

489 46250 3.5 10.2 9.9 0.7

The peak area given for each fragment was the average of 18 analyses.

than the uncertainty of the pUC19 calibrant from the digital PCR measurement (12%, see Table 2) indicating that amplicons quantified in this way may be suitable as secondary reference materials since the HPLC measuring system resulted in only a slight increase in uncertainty. Measurement of TLX3 304 bp Amplicon Concentration by IDMS. Validation of measuring systems is contextdependent, and in this case identification of potential biases in measurement was the major issue since interference, limit of detection, and robustness had little impact on the measuring systems. The HPLC measuring system quantified the intact amplicon, whereas the IDMS measuring system quantified deoxynucleotide monophosphates after enzymatic hydrolysis of the amplicon; the HPLC measuring system was calibrated with pUC19 MspI digest that had an assigned value from dPCR analysis, whereas the IDMS measuring system was calibrated with gravimetrically prepared mixtures of deoxynucleotide monophosphates. Bias in the HPLC measuring system would be caused from unresolved materials with the same retention time as the amplicon and in the IDMS measuring system from incomplete hydrolysis or inaccurate preparation of calibration standards. The IDMS measurement of the 304 bp amplicon was calculated as the average of the four concentration values, one from each of the four nucleotides, calculated using eq 3. Comparison to the HPLC measurement is illustrated in Figure 3 and shows that despite complete independence of the

analyte. Thus, selection of the pUC19 peak to serve as calibrant that is closest in size to the DNA amplicon to be measured may not be necessary. Measurement of TLX3 304 bp Amplicon Concentration by HPLC. A target sequence with high GC content was used to evaluate the HPLC measuring system since qPCR measurements of high GC regions may be highly variable.8 The measurand, i.e., the quantity intended to be measured,26 was a 304 base pair sequence of the promoter region of the human gene TLX3 that has a GC content of 73% and is commonly methylated in pediatric pre-B cell acute lymphoblastic leukemia.14 An amplicon corresponding to this sequence was prepared using end-point PCR (primers given in the Supporting Information) and purified by ion-exchange HPLC. The amplicon was quantified by HPLC using the dPCR measured pUC19 solution as calibrant. Measurements were performed by bracketing five replicate analyses of the amplicon with five replicate analyses of the calibrant pUC19 solution. The 304 bp amplicon has a calculated MR/(ε × 10−3) of 38.7, and this is closest to that of the 147 bp pUC19 fragment’s of 38.8. The difference between these values is 0.2%, and this means that the peak area for the 304 bp amplicon would be 0.2% smaller than an equal mass of the pUC19 147 bp fragment. The pUC19 fragment that was most similar in size was the 331 bp fragment, and in this case the difference in MR/ (ε × 10−3) values was 39.0−38.7 = 0.3 or 0.7%. The HPLC measured concentration may be corrected for this bias, or the uncertainty of the concentration measurement may be expanded to include this bias. If the concentration is corrected for the bias due to differences in MR/ε, then the HPLC measurement uncertainty must include the uncertainty of the bias correction. The uncertainty of the value of this bias would depend on the uncertainty of the calculated molar absorption coefficients. Table 4 illustrates the differences in measured 304 bp amplicon concentration obtained when seven different pUC19 fragments were used as calibrants. The uncertainty was calculated as specified in materials and methods for each separate calibrant fragment. The average of the seven concentration measurements was 10.1 ng/μL with an RSD of 4.1%. This variability between values was much greater than that expected from differences in MR/ε so most likely reflects chromatographic factors such as resolution, peak shape, and adsorption effects. The standard uncertainty of the average of the seven concentration measurements, calculated as the square root of the sums of squares of standard uncertainties divided by the square root of n, was 0.7 ng/μL giving a relative expanded uncertainty (k = 2) of 14.0%. Thus, there was no advantage in using all seven measurements since the combined relative uncertainty was very similar to each of the individual measurements. The relative expanded uncertainty of the TLX3 concentration was thus 14%, and this value was only slightly higher

Figure 3. Comparison of HPLC and IDMS measurements of the 304 bp amplicon. The error bars represent twice the standard deviation of the measurements.

measuring systems there was striking concordance of concentration values. The concordance of the two independent measurements validates the approach of using dPCR measured pUC19 as a calibrant for quantifying DNA amplicons by HPLC. Contamination of PCR Master Mixes. At the 6 month stability check of the pUC19 candidate reference material, 1662

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reagents since none of the common manufacturers discloses this parameter.

positive amplification was consistently detected in the controls that had no added template DNA (NTCs) using the same assay reagents as for the initial measurements. The qPCR Ct values in the NTCs were similar to the test samples with added template DNA, and the NTC amplicons were not distinguishable on capillary electrophoresis from those obtained by amplification of the template DNA (Supporting Information Figure S-2). Despite implementation of rigorous quality control measures and sequential investigation of PCR reagents the background contamination was still present. Therefore, we suspected the PCR master mix was the most probable contamination source since several studies have shown that commercial PCR master mixes can contain traces of microbial DNA.27−31 Evaluation of PCR master mixes from four different manufacturers (Agilent, Roche, ABI, Qiagen, not in order) showed that only one resulted in negative NTCs (see Supporting Information Figure S-4). Figure 4 shows the effect of treating a suspect PCR master mix with DNase I (conditions in the Experimental Section)

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CONCLUSION We have shown that HPLC calibrated with dPCR measured DNA ladder can be used to quantify a DNA amplicon with high GC content and that the concentration value obtained from the HPLC measurement is concordant with the value from IDMS. Since the two measuring systems are completely independent and measurement biases are not correlated, then the HPLC measuring system has been validated for this amplicon. Application of this measuring system to other amplicons may be valid provided fractionation by ion-exchange HPLC is used for purification of crude PCR materials and that the HPLC fraction contains a single product. HPLC measurement of amplicon DNA quantity using dPCR measured calibrant is more cost-effective than IDMS and overcomes the problems that may be encountered with PCR measurement of high GC and methylated sequences as has been observed in clinical diagnostics.8 In addition, there is no requirement to develop and validate specific qPCR assays for each DNA amplicon to be measured since the same HPLC measuring system can be used for amplicons within the size range resolved by the HPLC column irrespective of nucleotide sequence. The bias that may be introduced due to a difference between MR/ε of analyte and calibrant is likely to be in the order of 1%. The dPCR assays used to measure the concentration of the MspI digest of pUC19 were designed to target those fragments most closely matching the lengths of amplicons prepared in this laboratory as putative DNA reference materials. The target fragments were from the ampicillin resistance gene and were found to be present in commercial master mixes. A procedure for identifying and removing the contamination was presented. HPLC is a very useful analytical tool for DNA quantification providing precision within 1.6% with high accuracy.12 A widely applicable DNA quantitative reference material with defined mass concentration, such as the pUC19 material used in this work, will facilitate the rapid characterization of biomarkerspecific genetic reference materials that would be difficult to characterize using dPCR such as those with GC-rich target regions. The biomarker-specific reference materials can then be used for reliable quantitative measurement of genetic biomarkers in biological samples that may then be traceable to the international system of units.

Figure 4. qPCR amplification curves for pUC19/MspI (nominal 800 copies/μL) using assay 1 with and without DNase I pretreatment of master mix. Assay 2 gave similar results shown in the Supporting Information. The blue horizontal line represents the detection threshold value.

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prior to addition of the DNA template as suggested by Furrer et al. and followed by others.32−34 Following DNase I pretreatment of the master mix, amplification was not observed in replicate NTCs, and the pUC19 solution had an average Ct value of 29.4. However, with no DNase I pretreatment, positive amplification was observed in replicate NTCs and the pUC19 solution had average Ct value of 28.3. Consistent with removal of background contamination during the DNase I pretreatment step, the Ct value for pUC19 amplified using DNase I treated master mix was higher than that obtained using nontreated master mix. Contamination of DNA polymerase with ampicillin resistance genes has been observed previously.18 Though several means to overcome the DNA contamination of Taq polymerase have been reported,34−37 there is still a need for increased awareness of possible DNA contamination in molecular biology

ASSOCIATED CONTENT

S Supporting Information *

Table S-1 listing pUC19 MspI restriction fragment properties, equation S-1 for converting copy number concentration to mass concentration, qPCR conditions, Table S-2 listing primers and probes for qPCR and dPCR assays, primers for preparation of TLX3 304 bp amplicon, concentration of dNMP standards used for IDMS, LC−MS parameters, Figure S-1 dPCR heat map, Figure S-2 qPCR curves, Figure S-3 qPCR curves, Figure S-4 qPCR curves. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 1663

dx.doi.org/10.1021/ac302925f | Anal. Chem. 2013, 85, 1657−1664

Analytical Chemistry

Article

Author Contributions

(21) Burke, D. G.; Griffiths, K.; Kassir, Z.; Emslie, K. Anal. Chem. 2009, 81, 7294−7301. (22) Reagent Kit Guide DNA 500 and DNA 1000 Assay; Agilent Technologies: Waldbronn, Germany, 2003. (23) Ellison, S. L. R.; Emslie, K. R.; Kassir, Z. Anal. Bioanal. Chem. 2011, 401, 3221−3227. (24) Nic, M.; Jirat, J.; Kosata, B. IUPAC, Compendium of Chemical Terminology, XML on-line corrected version, 2012 ed.; updated by A. Jenkins; Blackwell Scientific Publications: Oxford, U.K., 1997. http:// goldbook.iupac.org. (25) Tataurov, A. V.; You, Y.; Owczarzy, R. Biophys. Chem. 2008, 133, 66−70. (26) International Vocabulary of Metrology; ISO/IEC: Geneva, Switzerland, 2007. (27) Tilburg, J. J. H. C.; Nabuurs-Franssen, M. H.; Hannen, E. J.; Horrevorts, A. M.; Melchers, W. J. G.; Klaassen, C. H. W. J. Clin. Microbiol. 2010, 48, 4634−4635. (28) Tondeur, S.; Agbulut, O.; Menot, M. L.; Larghero, J.; Paulin, D.; Menasche, P.; Samuel, J. L.; Chomienne, C.; Cassinat, B. Mol. Cell. Probes 2004, 18, 437−441. (29) Newsome, T.; Li, B.; Zou, N.; Lo, S. C. J. Clin. Microbiol. 2004, 42, 2264−2267. (30) Grahn, N.; Olofsson, M.; Ellnebo-Svedlund, K.; Monstein, H. J.; Jonasson, J. FEMS Microbiol. Lett. 2003, 219, 87−91. (31) Philipp, S.; Huemerb, H. P.; Irschicka, E. U.; Gassnerc, C. Transfus. Med. Hemother. 2010, 37, 21−28. (32) Furrer, B.; Candrian, P.; Wieland, P.; Luthy, J. Nature 1990, 346, 324. (33) Rochelle, P.; Weightman, A.; Fry, J. BioTechniques 1992, 13, 520. (34) Heininger, A.; Binder, M.; Ellinger, A.; Botzenhart, K.; Unertl, K.; Döring, G. J. Clin. Microbiol. 2003, 41, 1763−1765. (35) Nilsen, I. W.; Øverbø, K.; Havdalen, L. J.; Elde, M.; Gjellesvik, D. R.; Lanes, O. PLoS One 2010, 5, e10295. (36) Mohammadi, T.; Reesink, H. W.; Vandenbroucke-Grauls, C. M. J. E.; Savelkoul, P. H. M. J. Microbiol. Methods 2005, 61, 285−288. (37) Rueckert, A.; Morgan, H. W. J. Microbiol. Methods 2007, 68, 596−600.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The commercial master mixes specified in this study cannot be recommended as free from or containing contaminating DNA as this is batch-dependent. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The sequence of the TLX3 promoter region used in this work was provided by N. Wong of the Murdoch Childrens Research Institute, Melbourne Australia. Some of this work was undertaken as part of a memorandum of understanding between the National Institute of Metrology of the People’s Republic of China and the National Measurement Institute, Australia and was funded both by the Ministry of Science and Technology of the People’s Republic of China, as a part of key project (no. 2008BAK41B01) in the National Science & Technology Pillar Program (L.D., W.J.) and by the Australian Government National Enabling Technology Strategy (S.B., K.R.E., L.P., M.F.-S., S.F., and D.B.).

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