Performance of high-throughput DNA quantification methods

Background

The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen^® assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses.

Results

The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures.

Conclusion

The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

     

Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion.

The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.     

Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma

A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.     

Assay for the quantification of intact/fragmented genomic DNA

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.     

Maximizing signal-to-noise ratio in the random mutation capture assay

The 'Random Mutation Capture' assay allows for the sensitive quantitation of DNA mutations at extremely low mutation frequencies. This method is based on PCR detection of mutations that render the mutated target sequence resistant to restriction enzyme digestion. The original protocol prescribes an end-point dilution to about 0.1 mutant DNA molecules per PCR well, such that the mutation burden can be simply calculated by counting the number of amplified PCR wells. However, the statistical aspects associated with the single molecular nature of this protocol and several other molecular approaches relying on binary (on/off) output can significantly affect the quantification accuracy, and this issue has so far been ignored. The present work proposes a design of experiment (DoE) using statistical modeling and Monte Carlo simulations to obtain a statistically optimal sampling protocol, one that minimizes the coefficient of variance in the measurement estimates. Here, the DoE prescribed a dilution factor at about 1.6 mutant molecules per well. Theoretical results and experimental validation revealed an up to 10-fold improvement in the information obtained per PCR well, i.e. the optimal protocol achieves the same coefficient of variation using one-tenth the number of wells used in the original assay. Additionally, this optimization equally applies to any method that relies on binary detection of a small number of templates.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。     

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.     

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.     

孓遗植物桫椤RAPD分析中主要反应参数的影响

为得到桫椤RAPD扩增的最佳条件,对影响桫椤RAPD扩增的模板纯化方法、模板含量、酶浓度、Mg²⁺浓度、dNTP浓度和引物浓度及扩增程序等多种因素进行了探讨.结果表明,用玻璃奶和蛋白酶K纯化后获得的DNA作为模板进行RAPD反应,其扩增带比用未纯化的和仅用无水乙醇再沉淀的DNA为模板更亮,更清晰,重复性更好;模板浓度、酶浓度、Mg²⁺及dNIT浓度、引物浓度及退火温度这些因素都会对RAPD产生影响.经过本实验的探索,发现桫椤RAPD扩增的最佳体系是(2.5×10^-2mL反应体积):模板浓度为70ng,Mg²⁺浓度为2.5mmol·L^-1,dNTP为0.2mmol·L^-1,引物3×10^-4mL;最佳扩增程序为:94℃200s,一个循环;94℃60s,36℃60s,72℃120s,40个循环:72℃600s,一个循环:4℃保存.     

Capillary DNA Sequencing: Maximizing the Sequence Output

Like most other DNA sequencing core facilities, one of our continuing goals is to improve our sequence output without substantially adding to cost. To minimize sample-to-sample variability in template DNA concentration, we implemented the rolling circle amplification (RCA) procedure for preparing our DNA templates. In addition to saving time and reducing the number of steps in template DNA preparation, the RCA method has the potential to normalize the DNA concentration in samples that can be sequenced directly without additional purification. In the present study, we used RCA-generated templates to test a recently reported procedure that increased sequence quality by resuspending the sequenced products in low concentrations of agarose before capillary electrophoresis (CE) on a MegaBACE 1000 platform. Although we did not obtain the expected result using the specified procedure, a modification resulted in up to 60% increase in total sequence yield per sample plate. A combination of agarose and formamide-EDTA in the resuspension solution enabled us to generate long-read and high-quality sequences for more than 38,000 templates with minimal additional cost.     

Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR

Marine experimental stem cell transplantations require the accurate discrimination and quantification of donor cells from host cells. A Y-chromosome-specific, quantitative real-time PCR (kinetic PCR) protocol for blood-derived DNA was developed. The assay sensitivity was extremely high with accurate detection of only 10 pg (six copies of Y target DNA) in a variable background of female DNA background ranging from 2.5 to 50 ng. The dynamic range of the assay provided accurate results ranging from 2.2 x 10(-2)% to 100% of male DNA in female background. The kinetic PCR assay can be used in all mouse strains, and a sample size as low as 2.5 ng total DNA is sufficient for analysis. Therefore, kinetic PCR allows engraftment kinetic studies on repeated blood draws of individual animals with no need for sacrifice. Compared to conventional PCR, the assay is much simplified, as neither the accurate adjustment of sample DNA concentration nor a post-reaction analysis procedure is required. The procedure is simple, free of radioactivity, and permits a throughput of 500-600 reactions per day.     

Simultaneous quantification of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS)

Cyclophosphamide is a cytotoxic prodrug with a very narrow therapeutic index. To study the clinical pharmacology of cyclophosphamide in a large cohort of patients a previously published method for the simultaneous quantitative determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS) was optimized. Addition of an isotopically labelled internal standard and adaptation of the gradient resulted in a fast, robust and sensitive assay. Because 4-hydroxycyclophosphamide is not stable in plasma, the compound is derivatized with semicarbazide immediately after sample collection. Sample preparation was carried out by protein precipitation with methanol-acetonitrile (1:1, v/v), containing isotopically labelled cyclophosphamide and hexamethylphosphoramide as internal standards. The LC separation was performed on a Zorbax Extend C18 column (150 mm x 2.1 mm ID, particle size 5 microm) with 1 mM ammonium hydroxide in water-acetonitrile (90:10, v/v) as the starting gradient, at a flow-rate of 0.40 mL/min with a total run time of 6 min. The lower limit of quantification (LLQ, using a 100 microL sample volume) was 200 ng/mL and the linear dynamic range extended to 40,000 ng/mL for cyclophosphamide and 50-5000 ng/mL for 4-hydroxycyclophosphamide. Accuracies as well as precisions were lower than 20% at the LLQ concentration and lower than 15% for all other concentrations. This method has been successfully applied in our institute to support ongoing studies into the pharmacokinetics and pharmacogenetics of cyclophosphamide.     

Validation of ELISA for the determination of anti-ricin immunoglobulin G concentration in mouse sera.

An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9+/-11.3%, 102.7+/-10.8%, 99.0+/-7.2%, and 95.9+/-11.3%, respectively (n=10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1+/-7.3%, 100.2+/-5.8%, and 103.7+/-8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed (n=7 x 73). The results indicated that the four-parameter logistic (4PL) equation, y=(a-d)/(1+(x/c)b)+d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.     

A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system

Various assays are available for quantification of DNA in solution, but none has been described that is both sensitive and specific for double-stranded (ds) DNA and features practical properties such as low dye and equipment costs, speed, and highly parallel microplate formats. Here we show that quantitative and sensitive measurement of ds DNA in solution is achieved using a 96-well microplate SYBR Green I assay and a standard uv transillumination-based gel-imaging system for detection. Specific detection of ds DNA was obtained over a broad concentration range of 0.5-500 ng using a single low dye concentration of up to 1/6250. Measured SYBR Green I fluorescence was not significantly affected by pH variation (4-10), assay volume (50-250 microliter l), and time (4-15 min), and measurements were appreciably compatile with commonly encountered concentrations of contaminating salts, organics, detergents, and other substances. ds DNA yielded up to 13-fold higher fluorescence compared to single-stranded DNA or RNA, but this ratio was dependent somewhat on GC content and fragment size. Of note, linear ds DNA fluoresced significantly stronger than supercoiled plasmid DNA. Our method should be broadly applicable for sensitive, rapid, and inexpensive ds DNA quantification in the average molecular biology laboratory.     

Characterization of <Emphasis Type="Italic">target</Emphasis> nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template

Faecal material has increasingly become an important non-invasive source of DNA for wildlife population genetics. However, DNA from faecal sources can have issues associated with quantity (low-template and/or low target-to-total DNA ratio) and quality (degradation and/or low DNA-to-inhibitor ratio). A number of studies utilizing faecal material assume and compensate for the above properties with minimal characterization of quantity or quality of target DNA, which can unnecessarily increase the risk of downstream technical problems. Here, we present a protocol which quantifies faecal DNA using a two step approach: (1) estimating total DNA concentration using a Picogreen^TM fluorescence assay and (2) estimating target nuclear DNA concentration by comparing amplification products of field samples at suspected concentrations to those of control DNA at known concentrations. We applied this protocol to faecal material collected in the field from two species: woodland caribou (Rangifer tarandus) and swift fox (Vulpes velox). Total DNA estimates ranged from 6.5 ng/μl to 28.6 ng/μl (X = 16.2 ng/μl) for the caribou extracts and 1.0–26.1 ng/μl (X = 7.5 ng/μl) for the swift fox extracts. Our results showed high concordance between total and target DNA estimates from woodland caribou faecal extracts, with only 10% of the samples showing relatively lower target-to-total DNA ratios. In contrast, DNA extracts from swift fox scat exhibited low target DNA yields, with only 38% (19 of 50) of the samples showing comparative target DNA amplification of at least 0.1 ng. With this information, we were able to estimate the amount of target DNA entered into PCR amplifications, and identify samples having target DNA below a lower threshold of 0.2 ng and requiring modification to genotyping protocols such as multiple tube amplification. Our results here also show that this approach can easily be adapted to other species where faeces are the primary source of DNA template.     

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid–liquid-extraction and the treatment with hydrogen peroxide for the 2–20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5–5 μg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.     

Determination of rimantadine in rat plasma by liquid chromatography/electrospray mass spectrometry and its application in a pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of rimantadine in rat plasma. Rimantadine was extracted by protein precipitation with methanol, and the chromatographic separation was performed on a C(18) column. The total analytical run time was relatively short (4.6 min), and the limit of assay quantification (LLOQ) was 2 ng/mL using 50 microL of rat plasma. Rimantadine and the internal standard (amantadine) were monitored in selected ion monitoring (SIM) mode at m/z 180.2 and 152.1, respectively. The standard curve was linear over a concentration range from 2 to 750 ng/mL, and the correlation coefficients were greater than 0.999. The mean intra- and inter-day assay accuracy ranged from 100.1-105.0% to 100.3-104.0%, respectively, and the mean intra- and inter-day precision was between 1.3-2.3% and 1.8-3.0%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in rats after oral administration of rimantadine hydrochloride at the dose of 20 mg/kg.