6 research outputs found
Evaluation of the relationship of physical linkage to genomic distance, using polysaccharide precipitation-extracted DNA.
<p>(<b>a</b>) In this analysis, FAM-labeled “mile marker” assays targeting sequences at different distances (1–210 kb) from the <i>RPP30</i> anchor sequence were paired with a HEX-labeled assay specific to the <i>RPP30</i> anchor sequence. Control assays utilized an anchor assay sequence in <i>EIF2C</i>, which resides on another chromosome. (<b>b</b>) The percentage of linked molecules at each genomic distance is shown as a function of distance. Means (of triplicate measurements) and 95% confidence intervals are shown.</p
Drop-Phase schematic.
<p>A genomic DNA sample is emulsified into aqueous droplets in an oil-aqueous reverse emulsion. Allele-specific fluorescence probes (FAM, blue; and HEX, green) are used to detect alleles at two different loci. Following PCR, the droplets are positive for one fluorophore (blue or green), positive for both fluorophores (orange), or positive for neither fluorophore, depending on the alleles they contained at the beginning of the reaction. (<b>a</b>) <i>Trans</i>-configured alleles partition independently into droplets. Co-partitioning (orange) is therefore governed by chance. (<b>b</b>) <i>Cis</i>-configured alleles tend to co-segregate into the same droplets, because they are physically linked; co-partitioning greatly exceeds chance expectation. (<b>c</b>) Restriction digest at a site between the <i>cis</i>-configured alleles abolished co-partitioning of the two alleles; co-partitioning again occurs to the extent expected by chance.</p
Phasing <i>CFTR</i> variants in the genomes of cystic fibrosis patients.
<p>(<b>a</b>) Locations and genomic distances separating the variants along the <i>CFTR</i> gene in the tested cell lines. (<b>b</b>) Assembly of four duplex assays to redundantly evaluate phase of screened variants. (<b>c</b>) Physical linkage of <i>CFTR</i> variants as measured by Drop-Phase, as a function of genomic distance (horizontal axis). Blue diamonds: allele-pairs inferred to be <i>cis</i>-configured; purple squares: allele-pairs inferred to be <i>trans</i>-configured. The black line is an exponential curve fit to the <i>cis</i>-configured allele-pairs. Four duplex assays were performed per variant pair. Variants were classified as <i>cis</i>- or <i>trans</i>-configured based on measured positive linkage or lack of linkage, respectively. Samples were analyzed in duplicate.</p
Haplotypes formed by <i>CFTR</i> variants in six cystic fibrosis patients.
<p>Key: Hap 1 = Haplotype 1, Hap 2 = Haplotype 2</p><p>Haplotypes formed by <i>CFTR</i> variants in six cystic fibrosis patients.</p
Multiplexed Target Detection Using DNA-Binding Dye Chemistry in Droplet Digital PCR
Two years ago, we described the first
droplet digital PCR (ddPCR)
system aimed at empowering all researchers with a tool that removes
the substantial uncertainties associated with using the analogue standard,
quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis
probe-based assays for the absolute quantification of nucleic acids.
Due to significant advancements in droplet chemistry and buoyed by
the multiple benefits associated with dye-based target detection,
we have created a “second generation” ddPCR system compatible
with both TaqMan-probe and DNA-binding dye detection chemistries.
Herein, we describe the operating characteristics of DNA-binding dye
based ddPCR and offer a side-by-side comparison to TaqMan probe detection.
By partitioning each sample prior to thermal cycling, we demonstrate
that it is now possible to use a DNA-binding dye for the quantification
of multiple target species from a single reaction. The increased resolution
associated with partitioning also made it possible to visualize and
account for signals arising from nonspecific amplification products.
We expect that the ability to combine the precision of ddPCR with
both DNA-binding dye and TaqMan probe detection chemistries will further
enable the research community to answer complex and diverse genetic
questions
High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number
Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics