Biomedical subjects

F Oaks

Publications and source records attributed to F Oaks.

5 recordsLinked to original sources

PubMed2000

DNA sequencing in a monolithic microchannel device.

We present 50 cm long microchannels in a monolithic device for high resolution, long read-length DNA sequencing. These devices were fabricated and bonded in borofloat glass using unconventional photolithography techniques with 48-188 independent, straight microchannels. The microchannel DNA separation was tested with POP-6 polymer and a DNA sequencing ladder separated at room temperature and 200 V/cm. Single-base resolution greater than 600 bases was achieved and the sequence base called to 640 bases with 98% accuracy. Under the same experimental conditions, the performance of the microchip was identical to a fused-silica capillary with similar cross-sectional area.

Animals↗

PubMed1999

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.

Buffers↗

PubMed1998

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.

Alleles↗

PubMed1998

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.

Chromosome Mapping↗

PubMed1997

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.

Algorithms↗