Rapid Detection and Identification of Hepatitis C Virus (HCV) Sequences using Mismatch-Tolerant Hybridization Probes: A General Method for Analysis of Sequence Variation

Pierce, Kenneth E.; Wangh, Lawrence J.

doi:10.2144/000114076

Cited by 3 publications

(2 citation statements)

References 16 publications

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“…It generates high concentrations of single-stranded DNA that can be analyzed at the end point using probes which hybridize over a wide temperature range [65].…”

Section: Late-pcrmentioning

confidence: 99%

The Polymerase Chain Reaction

Sensabaugh¹,

Beroldingen²

2019

Foresic DNA Technology

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Polymerase chain reaction (PCR) is an efficient and one of the most common methods used in biological sciences for in vitro multiplication of a target DNA molecule. The technique has significantly contributed in changing and developing different fields of biological sciences since 1980s. PCR has a vital role in supporting the processes involved in genetic engineering, particularly the cloning of DNA fragments used to modify the genomes of microorganisms, animals, and plants. Consequently, the technique has numerous applications in fundamental and applied research in medicine agriculture, environment, and bio-industry. The main focus of this chapter is to describe briefly the principles, methodology, various types, and applications of PCR in different fields. Besides, different components of PCR, trouble shooting during the execution, and limitations of the techniques are also outlined.

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“…It generates high concentrations of single-stranded DNA that can be analyzed at the end point using probes which hybridize over a wide temperature range [65].…”

Section: Late-pcrmentioning

confidence: 99%

The Polymerase Chain Reaction

Sensabaugh¹,

Beroldingen²

2019

Foresic DNA Technology

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“…Each probe is mismatch tolerant and can be used to distinguish >10 possible variants over a detection temperature of at least 20°C (Pierce and Wangh 2014). Since each probe in a set has its own pattern of increasing or decreasing T m s to its own regional variants, sets of 2, 3, …n Lights-On/Lights-Off probes can potentially distinguish >10 n related target sequences.…”

Section: Lights-on/lights-off Probesmentioning

confidence: 99%

Closed-Tube Barcoding

Sirianni

Yuan

Rice

et al. 2016

Genome

Self Cite

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Abstract:Here, we present a new approach for increasing the rate and lowering the cost of identifying, cataloging, and monitoring global biodiversity. These advances, which we call Closed-Tube Barcoding, are one application of a suite of proven PCR-based technologies invented in our laboratory. Closed-Tube Barcoding builds on and aims to enhance the profoundly important efforts of the International Barcode of Life initiative. Closed-Tube Barcoding promises to be particularly useful when large numbers of small or rare specimens need to be screened and characterized at an affordable price. This approach is also well suited for automation and for use in portable devices.

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Low-Concentration Initiator Primers Improve the Amplification of Gene Targets with High Sequence Variability

Pierce

Wangh

2015

Methods in Molecular Biology

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The amplification and detection of diverse strains of an infectious virus or bacteria, or variants within a gene family is important for both clinical and basic research but can be difficult using conventional PCR. This report describes and illustrates a novel closed-tube method for amplifying and characterizing heterogeneous target sequences using members of the CTX-M beta-lactamase gene family. Different subgroups of CTX-M genes exhibit low sequence identity, but accurate and efficient detection of these variants is critical because they all confer resistance to penicillin, cefotaxime, and other antibiotics of the beta-lactam class. The method combines a single pair of "thermodynamic consensus primers" (tcPrimers) with one or more "initiator primers" (iPrimers), added at low concentration (5-10 nM). Each iPrimer improves the initial amplification of one or more variants because it has fewer mismatches to its intended target than the more abundant tcPrimers. As a result of initial amplification, each heterogeneous sequence is shifted stepwise toward a better match with the tcPrimers. As soon as the tcPrimer hybridization takes place, amplification proceeds with high efficiency. The tcPrimer pairs can be designed for symmetric PCR or for Linear-After-The-Exponential (LATE)-PCR. LATE-PCR offers the advantage of generating single-stranded DNA that can be characterized for different gene variants in the same closed tube, using low-temperature mismatch-tolerant fluorescent probes.

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Rapid Detection and Identification of Hepatitis C Virus (HCV) Sequences using Mismatch-Tolerant Hybridization Probes: A General Method for Analysis of Sequence Variation

Cited by 3 publications

References 16 publications

The Polymerase Chain Reaction

The Polymerase Chain Reaction

Closed-Tube Barcoding

Low-Concentration Initiator Primers Improve the Amplification of Gene Targets with High Sequence Variability

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