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Blood test for cancer biomarkers using an electrochemical clamp assay

first_img Journal information: Nature Chemistry Most methods of studying cell-free nucleotides involve DNA sequencing or PCR studies. Both of these have drawbacks. DNA sequencing is expensive and patients often have to wait several weeks for results. PCR requires extensive sample preparation and modifications to make it sufficiently selective for point mutations. To improve upon these techniques, Jagotamoy Das, Ivaylo Ivanov, Laura Montermini, Janusz Rak, Edward H. Sargent, and Shana O. Kelley from the University of Toronto and Montreal Children’s Hospital have developed a method that selectively identifies mutations common in lung and skin cancer with little-to-no sample preparation. Their work appears in Nature Chemistry.Over the past two decades, cancer genomics has made great strides in identifying genetic markers for certain cancers. Two examples used in the current study are the KRAS and the BRAF markers. KRAS has several mutations associated with it that are indicators of lung, colorectal, and ovarian cancer. BRAF mutations are most notably associated with melanoma, the most deadly form of skin cancer. Many genetic cancer markers involve a point mutation on a particular gene. Point mutations are difficult to detect because they are in such small quantities in blood compared to normal cell-free nucleic acids, or wild type. One way that scientists have made PCR more sensitive is by using genetic “clamps” called Peptide Nucleic Acids (PNAs). These are strands of a complementary nucleotide sequence that bind to wild type sequences and consequently, amplifying the target sequence. Das, et al. combined this PNA clamp technique with an electrochemical probe to make a fast, selective, and cost-effective sensor. The first designed a clamp system to select for particular mutations in the KRAS gene. KRAS has seven mutations that are associated with lung cancer. Their model system included clamps for all of the other possible mutations except for 134A, their target mutation, and it had clamps form wild type sequences. DNA mutations get harder to hide Citation: Blood test for cancer biomarkers using an electrochemical clamp assay (2015, June 10) retrieved 18 August 2019 from Explore further Credit: Wikimedia Commons © 2015 More information: “An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum” Nature Chemistry, (2015) DOI: 10.1038/nchem.2270AbstractThe analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients. They designed an electrochemical probe functionalized with complementary nucleic acid strands to various target sequences. The functionalized probe is a nanostructured microelectrode made from using nanolithography to insert palladium-coated gold deposits on silicon-coated wafers. The nanostructured microelectrodes were functionalized with PNAs that were specific to the 134A mutation sequence. Once the target sequence binds to the probe, it is read using an electrocatalytic reporter pair (Ru(NH3)63+ and Fe(CN)63-). Based on the electrochemical signal, their probe selectively bound the 134A mutation sequence without binding wild type sequences.To optimize their clamp assay, Das et al. tested whether their system could detect all seven mutations in KRAS in a non-purified, complex mixture of complementary mutant target, non-complementary mutants, the wild-type sequence, total human RNA and the clamp cocktail. They found that all seven mutations can be selectively detected using their system based on which PNA is omitted from the mixture.The next step was to see if the wild type PNA clamp needed to be included in the system for selective detection of point mutations. They tested a mutant positive patient sample and a healthy donor sample, and tested with the wild type clamp included and excluded. The authors determined that without the wild type clamp, they could not achieve sensitivity at the desired levels. They then evaluated the sensitivity of their clamp assay by looking at the electrochemical signal relative to varying concentrations of RNA in the solution. They found a limit of detection of 1 fg μl-1 and a turnaround time of five minutes. Finally, to demonstrate that this clamp assay was applicable to other mutations, they tested for biomarkers in the BRAF gene. The BRAF studies worked similarly to the KRAS studies, demonstrating the general applicability of this technique.After establishing the integrity of their clamp assay system, Das et al. then tested whether their system could detect cell free nucleic acids from serum retrieved from patients with lung cancer and patients with melanoma. These samples were compared to healthy donors and validated using an established PCR clamp method. Importantly, they tested both purified samples and samples taken directly from the patients without any purification. Their clamp assay identified 3/14 lung cancer patients as positive for KRAS and 4/9 melanoma patients as positive for BRAF in both the purified and non-purified samples.This work illustrates a new method for detecting cell free nucleic acids using peptide nucleic acid clamps and a nanostructured microelectrode chip. It is sufficiently sensitive and selective to detect cancer biomarkers in patient blood. Compared to other methods it is cost effective, minimally invasive, and requires little sample preparation. (—Researchers have found an innovative way to detect cancer biomarkers in a person’s blood. Nucleic acids, the components of DNA and RNA, are typically located within the cell. However, sometimes these nucleic acids can be found circulating in the blood. Cancer patients tend to have more of these cell-free nucleic acids in their blood. A small portion of these cell-free nucleic can contain mutations associated with certain cancers. This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.last_img read more

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The legacy left behind

first_imgSitaron ke Paas – Kalpana Chawla, a biographical play is being organised in the Capital by Art Karat and Arpana Trust. The play, directed and conceived by Sushma Seth,  is based on the life of  the astronaut Kalpana Chawla, her aspirations, her fearless spirit, and the exceptional qualities of perseverance. It brings forth the legacy she leaves behind after her death.  The play is produced by Sushma Aggarwal and Krisna Shroff. The script is written by Nigam Prakash, Prem Sarin and Seth herself. Seth has been organising production oriented drama workshops for the last 12 years. Her last play was Rabindranath Tagore’s Tasher Desh. Seth was awarded the Kalpana Chawla Award for Excellence in 2012 which inspired her to write and direct a play on Chawla’s life. When: Today Where: Shri Ram Centre, Mandi House Timing: 6.30 pmlast_img read more

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3D Scanner Makes Inventory Photography More Efficient

first_imgFebruary 3, 2012 Does the never-ending snapping and posting of inventory photos on your web shop leave you feeling like you’re running a still-life photography studio? It may be time to ditch the digital camera. Last year, printer giant HP shipped the TopShot, a web-enabled laser printer that doubles as a 3-D scanner capable of capturing high-quality images of small objects. And while the tool is not perfect, our tests made it clear that the TopShot could streamline online inventory photography.What it does: The TopShot, which costs about $400, can scan any object that fits inside the printer’s 8½-by-11-inch scanning area. The image is captured with a digital camera mounted in a pivoting light-enabled stand. Prop up the stand, place your object in the scanning area, click a few buttons, and the scanner goes to work, taking photos from multiple angles. Users can print the resulting image or send it to a PC.What works: HP deserves credit for making a fully capable 3-D web printer that scans, prints and copies. We found product images to be surprisingly clean, well-lit and professional-looking. Business-ready apps are available, including support of storage tools like Google Apps or Setup is also easy: Just plug in the USB connection and follow the instructions. No separate software disks or drivers are needed.What could use some work: The TopShot is far too slow to be a commercial-grade scanner or printer. It will take you about a full minute to set up, scan and capture an image; cataloguing a large inventory of photos could require a significant time investment. You will also need to tinker with the software to get the perfect exposure for some objects. (Bright spots on shiny objects are a challenge.) And remember, products must be small enough to fit inside the scanning area–even a pair of adult shoes is too big. Additionally, replacement toner cartridges are not cheap: $50 for black-and-white; $56 for color. Imaging drums run another $80.Bottom line: For an e-tail or eBay business, the TopShot can be an intriguing way to make goods more enticing to customers.  Register Now » 2 min read Attend this free webinar and learn how you can maximize efficiency while getting the most critical things done right. Free Webinar | Sept 5: Tips and Tools for Making Progress Toward Important Goals This story appears in the February 2012 issue of . Subscribe »last_img read more