Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP

Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing‐based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost‐effective DNA length measurement for STRs and resequencing of small DNA fragments.     

Locus-specific brackets for reliable typing of Y-chromosome short tandem repeat markers

Short tandem repeat (STR) loci, widely used as genetic markers in disease diagnostic studies and human identity applications, are traditionally genotyped through comparison of allele sizes to a sequenced allelic ladder. Allelic ladders permit a floating bin allele calling method to be utilized, which enables reliable allele calling across laboratories, instrument platforms, and electrophoretic conditions. Precise sizing methods for STR allele calling involving fixed bins can also be used when a high degree of precision has been demonstrated within an instrument platform and a set of electrophoretic conditions. An alternative method for reliable genotyping of STR markers, locus-specific brackets (LSBs), is introduced here. LSBs are artificial alleles created through molecular biology manipulations to be shorter or longer than alleles commonly seen in populations under investigation. The size and repeat number of measured alleles are interpolated between the two LSB products that are mixed with the polymerase chain reaction-amplified STR alleles. The advantages and limitations of the LSB approach are described along with a concordance study between the LSB typing approach and other STR typing methods. Complete agreement was observed with 162 samples studied at 5 Y-chromosome loci.     

An allelic ladder based upon reference alleles for mtDNA SNP analysis using the SNaPshot technique

The analysis of mitochondrial DNA (mtDNA) single-nucleotide polymorphisms (SNPs) using the SNaPshot technique (Applied Biosystems) is a fast and sensitive method for the reliable identification of disease-associated mtDNA SNPs, genetic ancestry mtDNA SNPs and forensically important mtDNA SNPs. The detection of many SNPs in one multiplex PCR and one subsequent multiplex minisequencing reaction is challenging for laboratories who want to establish this technique, due to the problem that there is no allelic ladder available for mtDNA SNP analysis via SNaPshot technique. Normally, the laboratory has to invent long-term testing and studies. The interpretation of false and correct alleles is up to some specialists knowing the expected and the estimated size of each allele SNP. We here present a protocol to assemble up to 84 alleles of 42 different mtDNA SNPs in an allelic ladder that is based upon reference alleles. We recommend using allelic ladders/reference alleles for SNP analysis to maintain high-quality analysis standards.     

Precision and accuracy in fluorescent short tandem repeat DNA typing: assessment of benefits imparted by the use of allelic ladders with the AmpF/STR Profiler Plus kit

Base-calling precision of short tandem repeat (STR) allelic bands on dynamic slab-gel electrophoresis systems was evaluated. Data was collected from over 6000 population database allele peaks generated from 468 population database samples amplified with the AmpF/STR Profiler Plus (PP) kit and electrophoresed on ABD 377 DNA sequencers. Precision was measured by way of standard deviations and was shown to be essentially the same, whether using fixed or floating bin genotyping. However, the allelic ladders have proven more sensitive to electrophoretic variations than database samples, which have caused some floating bins of D18S51 to shift on occasion. This observation prompted the investigation of polyacrylamide gel formulations in order to stabilize allelic ladder migration. The results demonstrate that, although alleles comprised in allelic ladders and questioned samples run on the same gel should migrate in an identical manner, this premise needs to be verified for any given electrophoresis platform and gel formulation. We show that the compilation of base-calling data is a very informative and useful tool for assessing the performance stability of dynamic gel electrophoresis systems, stability on which depends genotyping result quality.     

Sizing short tandem repeat alleles in capillary array gel electrophoresis instruments.

A 96-capillary array gel electrophoresis Applied Biosystems 3700 instrument has been used to analyse AMPF/STR SGM Plus short tandem repeat (STR) loci for forensic applications. This multiplex consists of ten STR loci plus the Amelogenin locus and currently forms the basis of the UK National DNA database that currently holds more than 1 million profiles. Of particular interest is the accuracy of allele designation that is determined by comparison with standard control allelic ladder markers. Some loci have higher standard deviations than others. In particular the high-molecular-weight HUMFIBRA alleles have high standard deviations of the order of 0.15 and it is these alleles that are most likely to be misdesignated. However, this risk is minimised by the analysis of at least five different allelic ladders across the array to estimate the mean size of each allele. In conjunction with this, a series of guidelines that can be programmed into expert systems are used to minimise risks of misdesignation. The efficacy of the procedures utilised are tested by computer simulation and demonstrated to be robust.     

Heteroduplex analysis of minisatellite variability

Minisatellites are tandem repeat arrays of middle size (5-100 bp) repeat units widely distributed in eukaryotic genomes. They have been related to several important features of human genome biology, including gene regulation, chromosomal fragile sites, and imprinting. In this report, we have critically assessed and employed heteroduplex analysis (HA) for the identification of different human minisatellite MsH43 alleles. This minisatellite is organized as a repeat array of 5-6 bp units spanning 0.5 kbp. Our results demonstrate that this procedure is an easy, rapid, and reliable method to document allelic diversity for this locus. This work suggests that HA will also be a useful tool for studying the polymorphism of other minisatellites with small repeat units.     

DNA ladders can be used to size polyphosphate resolved by polyacrylamide gel electrophoresis

PAGE is often used to resolve inorganic polyphosphates (polyP), but unfortunately polyP size ladders are not commercially available. Since several dyes that are commonly used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially available DNA size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available DNA ladders and these polyP fractions were then subjected to PAGE to determine the relationship between migration of DNA vs polyP, which was found to be: log₁₀(dsDNA length in bp) = 1.66 × log₁₀(polyP length in phosphate units) − 1.97. This relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that DNA size ladders can readily be employed to estimate polyP polymer lengths by PAGE.     

A comparative study of standards for determinations of trace wear metals in jet engine oils

The feasibility of using acid-treated metal powder standards for the determination of trace wear metals in spent jet engine oils is evaluated. The addition of 2:3:3 (v/v/v) HF—HCl—HNO₃ mixture to used lubricating oils did not cause any statistically reliable differences from results obtained with organometallic standards. Two sets of six correlation samples, one set being acid-treated, were analyzed for Cu, Cr, Mg, Fe, Mo and V by flame atomic absorption and flame atomic fluorescence spectrometry against each set of standards.     

Determination of indomethacin in serum and urine by electron-capture gas-liquid chromatography.

A specific and sensitive method for the quantitative determination of indomethacin in serum and urine is described. The drug is extracted at pH 5.0 with 1,2-dichloroethane and a portion of the organic extract is concentrated and made to react with diazoethane in diethyl ether. The ethyl ester derivative is analyzed by electron-capture gas-liquid chromatography, quantitation being achieved by comparison of peak areas for samples and standards, which are prepared in serum or urine and treated in the same manner as the samples. The limit of sensitivity is 50 ng/ml and the relative standard derivation for repeat determinations on the same sample is about 3%.     

Molecular weight determination of bulk polymer surfaces by static secondary ion mass spectrometry

The determination of molecular weights at surfaces of bulk polymer materials can be accomplished by static secondary ion mass spectrometry (SIMS) via fragments originating from repeat units and end groups. The intensity ratio of these fragments depends on the polymer chain length as seen for bisphenol-A-polycarbonate and perfluorinated polyethers (Krytox). A kinetic model of fragment ion formation explains the molecular weight dependent fragment intensities and links them to properties of the molecular weight distribution. In the most simple case one obtains the number average molecular weight <M_n> at the surface. This technique can be used for the determination of the molecular weight at bulk polymer surfaces such as a CD-ROM made from polycarbonate by injection molding.     

Flow injection analysis for calcium in serum,water and waste waters by spectrophotometry and by ion-selective electrode

The Flow Injection technique is shown to provide fast, reliable and sensitive methods for the determination of calcium in various aqueous as well as serum samples; spectrophotometric or potentiometric detection can be used. At sampling rates of 100–110 samples per hour, with 30-μl sample injections, high reproducibility of measurement and low reagent consumption are achieved in both methods. In the spectrophotometric method, the analytical readout is available within 12 s after sample injection at a total reagent consumption of 0.75 ml per analysis. The potentiometric measurement of the calcium activity in serum is placed on a reliable basis by alternating measurements of serum samples and aqueous standards without incurring any non-reproducible changes in potential between aqueous and serum solutions. This permits the simultaneous determination of pH and pCa, the analytical readout being available within XXX s of sample injection. The good agreement between the results obtained with the Flow Injection method and those attained by atomic absorption and EDTA titrations as well as pCa stat-measurements show that the new methods are potentially suitable for routine analysis.     

Electrochemical detection of DNA triplet repeat expansion

Hereditary neurodegenerative diseases are connected with the expansion of trinucleotide repetitive sequences in genomic DNA. Molecular diagnosis of these diseases is based on the determination of the triplet repeat length. Currently used methods involve PCR amplification followed by electrophoretic determination of the amplicon size. We propose a novel electrochemical technique based on hybridization of target DNA (tDNA) immobilized at magnetic beads with a reporter probe (RP) complementary to the triplet repeats (12 units per RP). The biotin-labeled RP is detected via an enzyme-linked electrochemical assay involving binding of streptavidin-alkaline phosphatase conjugate and transformation of electroinactive 1-naphthyl phosphate to electroactive 1-naphthol. Pyrimidine residues within sequences flanking the homopurine (GAA)n repeat in tDNA are premodified with osmium tetroxide, 2,2'-bipyridine (Os,bipy), introducing electroactive labels in tDNA. The length of the triplet expansion is calculated from the ratio of the intensities of electrochemical signals of hybridized RP/tDNA-Os,bipy. The normalized signal increases linearly with the repeat length between 0 and about 200 triplet units, allowing for discrimination between normal, premutated, and mutated alleles. Application of this method for the detection of the asymptomatic heterozygous carrier of expanded alleles is demonstrated.     

Identification of peaks in capillary zone electrophoresis based on actual mobilities.

A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.     

Reliable measurements of interfacial slip by colloid probe atomic force microscopy. I. Mathematical modeling

We developed a stable spread-sheet algorithm for the calculation of the hydrodynamic forces measured by colloid probe atomic force microscopy to be used in investigations of interfacial slip. The algorithm quantifies the effect on the slip hydrodynamic force for factors commonly encountered in experimental measurements such as nanoparticle contamination, nonconstant drag force due to cantilever bending that varies with different cantilevers, flattening of the microsphere, and calibration at large separations. We found that all of these experimental factors significantly affect the fitted slip length, approximately in the order listed. Our modeling is applied to fit new experimental data reproducibly. Using this new algorithm, it is shown that the fitting of hydrodynamic theories to experimental data is reliable and the fitted slip length is accurate. A "blind test" protocol was developed that produces a reliable estimate of the fitting error in the determination of both the slip length and spring constant. By this blind test, we estimate that our modeling determines the fitted slip length with an average systematic error of 2 nm and the fitted spring constant with a 3% error. Our exact calculation of the drag force may explain previous reports that the fitted slip length depends upon the shape and spring constant of the cantilever used to perform the measurements.     

Application of off-line size-exclusion chromatographic fractionation-matrix assisted laser desorption ionization time of flight mass spectrometry for proanthocyanidin characterization

In this work, we wanted to devise a reliable method to characterize polymerized forms of tannins, their structural information and mass distribution. Size-exclusion chromatography (SEC) is a chromatographic technique used to determine molecular mass (weight) distributions of polymers. One important step in the data treatment is the modeling of the calibration curves. Polystyrenes (PS) are standards usually used because no commercial procyanidin (PC) standards are available. An off-line coupling of SEC and MALDI was carried out to measure differences between polystyrenes and procyanidins. Thus, a new calibration curve was established; from 1000 to 8000 Da, there is a good correlation between the MALDI and PS calibration curves, in this field the PS calibration is correct and enables true mass determination. For masses above 8000 Da, PS calibration overestimates the real molecular weight of PC, overestimation of 53%. And for masses below 1000 Da, PS calibration underestimates their real molecular weight (10-15%). This means that to truly characterize PC, calibration based on PC standards is required.     

Development of certified reference materials of high-purity volatile organic compounds: purity assay by the freezing-point depression method

For accurate measurement of concentrations of substances by instrumental analysis, reliable calibration standards are needed. In Japan, national reference materials are supplied under the national standards dissemination system named the Japan Calibration Service System (JCSS). In JCSS, calibration standards for the analysis of environmental pollutants are supplied. For the traceability to the SI of reference materials for calibration in JCSS, the National Metrology Institute of Japan (NMIJ) is developing high-purity reference materials of volatile organic compounds (VOCs) as NMIJ CRMs. The freezing-point depression method, which has potential as a primary method of measurement, is employed for the determination of property value. In this paper, a development scheme of certified reference materials of high-purity VOCs is described. Presented at BERM-11, October 2007, Tsukuba, Japan.     

Using additional standards for increasing the accuracy of quantitative chromatographic analysis

Uncontrolled partial losses at the step of sample injection into a gas chromatographic column increase errors of determination by the external standard, absolute calibration, and standard addition methods. Modified method is proposed for quantitative analysis; it includes the introduction of additional standards into test samples and calculations by the ratio between the areas of chromatographic peaks and peaks of standards rather than the absolute areas of chromatographic peaks. The calculation equations are presented for modified methods of quantitative analysis using additional standards, including those for estimating random errors of determination. The relative standard deviations of peak areas were shown to be 6–38-fold lower than the analogous statistical characteristics of absolute areas. This ensures a high accuracy of quantitative determinations even under the conditions of low reproducibility of sample dosing. Solvents contained in the analyzed samples can be used as additional standards. This version can be recommended as a routine method of data representation and processing.     

Novel Sampling for the Determination of Naphthalene in Air by Gas Chromatography–Mass Spectrometry

Here are reported two new sampling method approaches for the determination of naphthalene in ambient air for concentrations from 0.25 to 18.7?µg/L. The first method used for gas phase naphthalene analysis produced an average recovery of 88.8% and the second method using headspace sampling produced an average recovery of 93.8%. The second method showed better recovery than the former, so it was used for subsequent comparative gas-phase determination of naphthalene. The second method was validated at various naphthalene concentrations and humidity using a naphthalene gas generator to produce various naphthalene standards and a naphthalene-monitoring instrument. The naphthalene concentrations generated using the gas generator and determined second sampling method with gas chromatography–mass spectrometry (GC–MS) were compared to the sensor measurements and were in good agreement. In summary, the sampling methods presented provided reliable gas-phase naphthalene determination when coupled with GC–MS.     

用电子探针定量分析超轻元素中的几个问题

碳，硼，氮及氧等元素在电子探针分析中常称之为超轻元素，这些元素的定量分析还存在许多包括测量和定量修正方面的困难。本文就质量吸收系数的不准确，超轻元素谱线的化学位移，及重元素的Ｌ，Ｍ及Ｎ系谱线对其干扰等方面的问题进行研究和讨论，并分别提出了解决这些问题的方法。选择合适的标样是获得准确定量分析结果的关键。     

A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp

Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards; these are usually manufactured from plasmid or viral DNA fragments, or more recently, from PCR products of defined sizes. We describe here the preparation of a molecular weight standard from a completely different DNA source - the uniquely organized genome of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150 and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications, including separation of PCR products and plasmid cloning targets. In addition, it is easy to prepare, inexpensive, and rivals the best of the commercial ladders.