High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

BACKGROUND: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. RESULTS: A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. CONCLUSIONS: Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability. We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation. RESULTS: SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe. CONCLUSIONS: Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring

Ratios of bottom meson branching fractions involving J/psi mesons and determination of b quark fragmentation fractions

We report a measurement of the ratios of the decay rates of the B^+, B^0 and B^0_s mesons into exclusive final states containing a J/psi meson. The final states were selected from 19.6 pb^{-1} of p-pbar collisions recorded by the Collider Detector at Fermilab. These data are interpreted to determine the bquark fragmentation fractions f_u, f_d and f_s. We also determine the branching fractions for the decay modes B^+ --> J/psi K^+, B^+ --> J/psi K^*(892)^+, B^0 --> J/psi K^0, B^0 --> J/psi K^*(892)^0 and B_s^0 --> J/psi phi(1020). We discuss the implications of these measurements to B meson decay models.Comment: 40 pages with 5 figures. Submitted to Phys. Rev. D. PostScript also available at http://www-cdf.fnal.gov/physics/pub96/cdf3609_bfrag_br_prd.p

The application of a predictive migration model for evaluating the compliance of plastic materials with European Food Regulations

A model for estimating the migration of organic substances from plastic materials into foodstuffs has been previously developed. The use of this model allows a shift away from expensive and time consuming migration analysis in foods to much simpler compositional analysis in the material. Starting with toxicologically-based specific migration values, like the over 400 specific migration limit values for organic substances listed in the European Union's Synoptic Document No. 7, the corresponding allowable maximum quantity limits of these substances in the plastic were calculated. The amount of migration that can occur depends on many different factors such as the natures of the plastic, substance and food, contact time and temperature conditions, material thickness and number of uses. These factors can result in a variety of situations which must be taken into account in the estimation of migration. The handling of the migration estimation model is demonstrated for several practical migr ation scenarios using a series of case studies

The application of a model for evaluating the compliance of plastic materials with food regulations

A model for estimating the migration of organic substances from plastic materials into foodstuffs has been previously developed. The use of this model allows a shift away from expensive and time consuming migration analysis in foods to much simpler compositional analysis in the material. Starting with toxicologically based specific migration values, like the over 400 SML values for organic substances listed in the European Union's Synoptic Document N. 7, the corresponding allowable limits (QM) of these substances in the plastic were calculated. The amount of migration that can occur depends on many different factors such as the natures of the plasic, substance and food, contact time and temperature conditions, material thickness and number of uses. These factors can result in a variety of situations wich must be taken into account in the estimation of migration. The handling of the migration estimation model is demonstrated for several practical migration scenarios using a series of ca se studies

Quantitative analysis of polymer additives by coupled SFE/SFC.

Supercritical carbon dioxide was used supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) to quantitatively analyze additives in polymers. A unique method was used to measure carbon dioxide flows at the system's outlets. Triacetine in Biopol and Irganox additives in Polypropylene were measured