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

Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4

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