Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

High-precision genotyping by denaturing capillary electrophoresis.

Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of +/-0.3 nucleotide are required to ensure with 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, operation at 60 degrees C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d'Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.

Functionalized tricarbocyanine dyes as near-infrared fluorescent probes for biomolecules.

The syntheses of three novel functionalized tricarbocyanine dyes are described. These dyes containing isothiocyanate and succinimidyl ester functional groups are reactive toward primary amines and can be used as fluorescent probes for biologically pertinent compounds such as amino acids and functionalized dideoxynucleotides. The absorption and fluorescence maxima occur in the near-IR region of the spectrum (770-810 nm). The succinimidyl ester proved to be very sensitive to hydrolysis and was generated in situ to label amino acids. The isothiocyanates were less susceptible to hydrolysis and were conjugated using organic modified [40% (v/v) acetonitrile] buffers to amino acids. A dye with an alkyl isothiocyanate moiety showed conjugation to amino-functionalized dideoxynucleotide triphosphates.

Improved single-strand DNA sizing accuracy in capillary electrophoresis.

Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.

New energy transfer dyes for DNA sequencing.

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.

New dye-labeled terminators for improved DNA sequencing patterns.

We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.

Flowable networks as DNA sequencing media in capillary columns.

A novel class of materials that self-assemble in water into equilibrium network structures with a well-defined mesh size consist of polyethylene glycols (PEG's) end-capped with micelle-forming fluorocarbon tails. These micellar systems form flowable aqueous gel-like networks that permit electrophoretic DNA sequencing in capillary columns. The gels have unusual rheological properties, including network breakdown under shear, resulting in plug flow that allows columns refill with complete ejection of byproducts of the previous sequencing analysis. In this system, DNA fragment electrophoretic mobilities are unaffected by the hydrophobicity of the polymer tails. Low molecular weight (M) PEG chains (M 8000) show catastrophic resolution loss for DNA fragments larger than 100 bases due to band broadening. For a longer PEG segment (M 35000) separating the end groups, band broadening occurs for DNA fragments larger than 300 bases, implying that the PEG segment length controls the mesh size in the equilibrium network structure. Optimum sequencing results were obtained from a 6% solution of a 1:1 mixture of C6F13 end-capped- and C8F17 end-capped PEG 35,000. The resolution limit of fluorescent-dye-labeled sequencing products in this formulation was 450 bases in 75 microns capillaries at 200 V/cm.

Quantitative analysis of DNA-sequencing electrophoresis.

At the heart of the DNA-sequencing process is a remarkably selective electrophoretic separation of up to 1000 oligonucleotide fragments, each differing in size by only a single nucleotide unit. A quantitative analysis of this separation is performed in terms of both selectivity and efficiency. It is shown that both the Ogston sieving and reptation migration mechanisms are operative. It is demonstrated that, under the conditions used in traditional sequencing electrophoresis, Joule heating does not significantly contribute to band broadening, and that diffusion is the primary contributor to plate height. An analytic expression is derived relating the peak width for each fragment to its molecular size. Calculations are presented showing that, when longer sequences are required, the maximum electrical field strength will be limited by the influence of biased reptation on the separation selectivity. Finally, it is shown that, when short sequences are required, the electrical field strength is limited by the ability to dissipate Joule heat, and that in these cases a tube format will be approximately 50% faster than a slab having a thickness equivalent to the tube diameter.

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing.

A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.

Validity testing of commercial urine cocaine metabolite assays: I. Assay detection times, individual excretion patterns, and kinetics after cocaine administration to humans.

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.

Validity testing of the TDx Cocaine Metabolite Assay with human specimens obtained after intravenous cocaine administration.

Clinical specimens obtained from human subjects after intravenous cocaine administration were analyzed by the TDx Cocaine Metabolite Assay (TDx) and by GC/MS for benzoylecgonine. The TDx results were significantly correlated with results by GC/MS assay with no evidence of bias in the TDx assay. All cocaine metabolite positive specimens (greater than or equal to 300 ng/ml) were confirmed by GC/MS. Detection times to the last positive specimen by TDx assay and GC/MS assay of four subjects after a 20-mg intravenous dose of cocaine ranged from 29.3 to 39.1 h and 27.9 and 36.6 h, respectively. Overall, the TDx assay was found to be highly specific and accurate for the detection and measurement of benzoylecgonine in urine.

Lack of validity of the KDI Quik Test Drug Screen for detection of benzoylecgonine in urine.

The validity of the Quik Test Drug Screen for detection of benzoylecgonine was assessed by testing, in random order under blind conditions, 50 aliquots of drug-free urine and 50 aliquots of urine containing 6000 ng/mL of benzoylecgonine. Three independent readers of the test results had an average overall accuracy of only 50%. Each specimen also was tested by TDx assay for cocaine metabolite. In contrast to the Quik Test, the TDx assay was completely accurate. The false positive and false negative rates of the Quik Test Drug Screen for benzoylecgonine in urine were found to be unacceptably high, and its use in urine testing for cocaine abuse is not recommended.