Strategies for Preventing Amplicon Contamination in Molecular Laboratory !

The Primer

The high sensitivity of the polymerase chain reaction (PCR)—theoretically with lower limits of detection as little as a single template molecule; practically, 10 to 100 copies for many assays as run—is one of its greatest strengths, but also its greatest weakness. As the method works through creating copies of its target, any positive sample can lead to large numbers of molecules which can in turn contaminate subsequent reactions and cause false positive results.

To get a sense of the scope of this, consider a successful “average” 25μl PCR somehow getting opened and spilled in the lab. This would contain on the order of 10^12 template copies (amplicons); in other words, if a thorough cleaning reduced this by a million fold, you’d still have a million amplicon copies “floating around,” each of which could contaminate a reaction. If you’re fortunate enough to have never experienced this first-hand, you can thank the widespread acceptance of real-time PCR methods, which do away with having to open reaction tubes post amplification, and perhaps gain an appreciation of why anyone who has been through the experience treats the risk as real and ever-present.

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Molecular Biology Videos: How to Perform Colony PCR!

This video on molecular biology demonstrates how to perform colony PCR as part of a cloning workflow using Thermo Scientific DreamTaq DNA Polymerase.

Republished for Medical Education, Awareness & Information

Source: ThermofisherScientific

PCR-Free Novel Genome Sequencing Technology

Will Nanopore Sequencing Make it Obsolete?

Genome sequencers have been tweaking polymerase chain reaction (PCR) amplification to avoid introducing artifacts into sequencing libraries, ranging from modifications in chemistries to the introduction of novel sequencing technologies that could obviate the need for PCR altogether.

PCR-related problems have included uneven amplification, causing overrepresentation of some sequence species and nucleotide misincorporation. In particular, sequencing genomes or genomic regions with extremely biased base composition remains a challenge to the currently available next-generation sequencing (NGS) platforms. These include, for example, the genomes of important pathogenic organisms like Plasmodium falciparum with a high adenine–thymine (AT) content and Mycobacterium tuberculosis with high guanine–cytosine (GC) content.

These genomes have proven difficult for sequencers because the standard library preparation procedures that employ PCR amplification cause uneven read coverage across these regions, leading to problems in genome assembly and variation analyses. 

Read more: PCR-Free Novel Genome Sequencing Technology

Disruptive sequencing based on nanopore technology has the potential to change
completely the way DNA sequencing is done.

Source: beawolf/Fotolia

Ebola Virus - Amplification-free direct detection on a hybrid optofluidic platform

Low-complexity detection of infectious diseases with high sensitivity and specificity is urgently needed, especially in resource-limited settings. Optofluidic integration combines clinical sample preparation with optical sensing on a single chip-scale system, enabling the direct, amplification-free detection of single RNA from Ebola viruses. The optofluidic system fulfills all key requirements for chip-based clinical analysis, including a low limit of detection, wide dynamic range, and the ability to detect multiple pathogens simultaneously.

Illustration of a virus and blood cells (Shutterstock)

Introduction

The recent Ebola and Zika outbreaks [1, 2] have made it clear that viral infections continue to pose diverse and widespread threats to humanity. Resource-limited settings, in particular, call for diagnostic devices and technologies that are robust and feature relatively low complexity for easy handling by potentially unskilled personnel. At the same time, such instruments need to fulfill all the technical requirements for accurate and reliable diagnosis. These include a limit of detection and dynamic range that are compatible with clinically observed viral loads as well as the ability to carry out multiplexed differential detection by screening simultaneously for several pathogens with similar clinical symptoms.

Figure 1. (a) Cross-sectional view of liquid-core ARROW. (b) Schematic of Automaton integrated with ARROW chip. (c) A hybrid optofluidic ARROW system. (d) Digitized fluorescence signal counts above background. (e) Concentration-dependent RNA counts for off-chip (open squares) and using the automaton (solid circles) sample preparation. Negative controls (SUDV, MARV) did not create any counts. Dashed line indicates predicted particle count determined from initial concentration and experimental parameters. EBOV, Zaire Ebola virus; MARV, Marburg virus; SUDV, Sudan Ebola virus. (Adapted from Cai et al., 2015 [12])

The 'gold standard' test for hemorrhagic fevers as well as other infectious diseases is real-time polymerase chain reaction (RT-PCR) [3]. PCR fulfills the sensitivity and specificity requirement for clinical testing. However, it is not ideal for resource-limited environments and point-of-care applications because of to its complexity. An alternative economic and portable option is antigen-capture enzyme-linked immunosorbent assay (ELISA) testing. However, ELISA requires more highly concentrated samples and thus its clinical application, especially for early disease detection, is restricted.

For the last two decades, the lab-on-chip approach, which features a small footprint and sample volume, has been considered as a promising candidate for the next generation low-complexity medical diagnostics [4]. Among all the approaches, optofluidics, which integrates optics and microfluidics in the same platform, has received increased attention [5, 6]. Microfluidics is ideal for performing biological sample processing on a chip-scale level and leads to miniaturization and simplification of the current diagnostic system. If it can be integrated with an optical sensing/read-out platform that enables high detection sensitivity down to the single pathogen level, an analytic system for which nucleic acid amplification is no longer needed becomes possible.

Figure 2. (a) Schematic of multi-mode interferometer (MMI) waveguide intersecting with liquid-core ARROW. (b) Excitation spots, 9 (blue), 8 (green), and 7 (red), generated by the MMI at wavelength of 488nm, 553nm and 633nm, respectively. (c) Optical signal detected from various labelled single virus particles. (Adapted from Ozcelik et al., 2016 [14])

In order to detect single molecular biomarkers and bioparticles, an in-flow based detection scheme is preferred. In a typical in-flow detection scheme, bioparticles are transported to the sensing region in a stream of gas or liquid where they are detected in transient fashion as they pass an optical interrogation region [7, 8]. Therefore, fast read-out of the optical signal from single bioparticles in sequence can be achieved, and many concerns associated with traditional surface-based sensing schemes such as unwanted nonspecific binding, probe photobleaching, and diffusion-limited transportation are eliminated.

Read more: Ebola Virus - Amplification-free direct detection on a hybrid optofluidic platform

Source: cli-online.com