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L Kotler

Publications and source records attributed to L Kotler.

10 recordsLinked to original sources
PubMed2000

DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Acrylic Resins↗
PubMed2000

Automatic comparisons of artificial digits never compared: learning linear ordering relations.

In 2 experiments, participants were trained to perform magnitude decisions, that is, decide which of 2 arbitrary symbols in a pair represented a larger magnitude. The symbols corresponded to locations on an implicit linear scale. Training resulted in a Stroop-like size congruity effect when the participants had to decide which symbol in a pair was physically larger. This effect, showing automaticity of the processing of magnitude relations, was also obtained for pairs never encountered during practice. The implications of these findings for processing of magnitude relations and for theories of automaticity are discussed.

Adult↗
PubMed1998

Routine DNA sequencing of 1000 bases in less than one hour by capillary electrophoresis with replaceable linear polyacrylamide solutions.

Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

Acrylic Resins↗
PubMed1998

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. A. Development of the cleanup protocol.

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.

Base Sequence↗
PubMed1998

A sample purification method for rugged and high-performance DNA sequencing by capillary electrophoresis using replaceable polymer solutions. B. Quantitative determination of the role of sample matrix components on sequencing analysis.

In the previous paper, a sample cleanup procedure for DNA sequencing reaction products was developed, in which template DNA was removed by ultrafiltration and the total concentration of salts (chloride and di- and deoxynucleotides) was decreased below 10 microM using gel filtration. In this paper, a quantitative study of the effects of these sample solution components on the injected amount and separation efficiency of the sequencing fragments in capillary electrophoresis is presented. The presence of chloride and deoxynucleotides in a total concentration above 10 microM in the sample solution significantly decreased the amount of DNA sequencing fragments injected into the capillary column. However, the separation efficiency was not affected upon increasing the amount of salt. On the other hand, in the presence of only 0.1 microgram of template in the sample (one-third of the lowest quantity recommended in cycle sequencing) and at very low chloride concentration (approximately 5 microM), the separation efficiency decreased by 70%, and the injected amount of DNA sequencing fragments was 40% lower compared to the sample cleaned by the new purification method. The deleterious effect of template DNA on the separation of sequencing fragments was suppressed in the presence of salt in a concentration above 100 microM in the sample solution. Separately, it was found that both the electric field strength and duration of injection affected the resolution of DNA sequencing fragments when the cleaned up sample solution was used. Separation efficiencies of 15 x 10(6) theoretical plates/m were achieved when the sample was loaded at low electric field, e.g., 25 V/cm for 80 s or less. The results demonstrate that the sample solution components (chloride, deoxynucleotides, template DNA) and injection conditions must be controlled to achieve high performance and rugged DNA sequencing analysis.

Chlorides↗
PubMed1998

Characterization of high molecular mass linear polyacrylamide powder prepared by emulsion polymerization as a replaceable polymer matrix for DNA sequencing by capillary electrophoresis.

In a previous paper, a 2% w/w replaceable high molecular mass linear polyacrylamide solution (high molecular mass LPA) was used to achieve long read-lengths for DNA sequencing by capillary electrophoresis (E. Carrilho et al., Anal. Chem. 1996, 68, 3305-3313). In that work, the polymer was prepared by polymerization in water at 6% w/w, followed by dilution to 2% w/w. In this study, an improved method for preparation of high molecular mass LPA was developed, based on inverse emulsion polymerization. With this polymerization procedure, the LPA results in a molecular mass of approximately 9 MDa, with characteristics of a fine powder of high purity and practically unlimited shelf life. Using size exclusion chromatography (SEC) and viscosity measurements to characterize the polymer, good batch-to-batch reproducibility was found. It was observed that the viscous polymer solutions made from these high molecular mass polymers require careful preparation and handling because the method of dissolution could affect the molecular mass distribution and the resultant separation of DNA components. Solutions containing 2% w/w of LPA made by emulsion polymerization were simple to prepare, resulting in excellent performance as a replaceable matrix for DNA sequencing by capillary electrophoresis. The viscosity of the polymer decreased exponentially when pressure was applied, allowing easy replacement from a capillary using a syringe. With a properly prepared matrix, a read-length of more than 1000 bases in 80 min with an accuracy better than 97%, and better than 99% for the first 800 bases, could be achieved.

Acrylic Resins↗
PubMed1994

Case study of the effects of prolonged and severe anorexia nervosa on bone mineral density.

Among the most severe sequelae of anorexia nervosa (AN) are its skeletal complications. Young women who have AN during adolescence may not attain their expected peak skeletal mass, and may enter adulthood with reduced bone mineral density (BMD) and an increased risk of fracture. This case history describes a young woman with severe AN that included prolonged exposure to both low body weight and amenorrhea. BMD measurement during the acute stage of her illness revealed severe osteopenia. Six years after recovery from AN, follow-up studies demonstrated only modest gains in BMD, with measurements for the hip and lumbar spine that are greater than 2 SDs below the age-matched mean.

Adult↗
PubMed1994

DNA sequencing: modular primers for automated walking.

Here we describe our progress in the development of the technology of DNA sequencing by primer walking based on the modular primer approach, which eliminates the primer synthesis bottleneck. Modular primers are assembled from 5-mers, 6-mers, or 7-mers selected from a presynthesized library of as few as 1000 oligonucleotides. This technology is expected to reduce the time per walk by a factor of 20 to 50, and the cost of DNA sequencing by a factor of 5 to 15. Both time and expense will be saved not only on the primer synthesis per se but, more importantly, as a result of the closed-end automation of the complete cycle of walking sequencing, where no human intervention will be required between the walks. Such closed-end automation has until now been precluded due to the need to synthesize a new primer for each walk. As a part of the closed cycle automation development, this report deals with modular primers used for sequencing with fluorescent dye terminators. The success rate and the quality of automated sequencing with modular primers are found similar to those with the conventional 17-20-base-long primers. One of the advantages of the described technique is simple purification without any proteins that need to be removed.

Base Sequence↗