scholarly journals A set of experimentally validated, mutually orthogonal primers for combinatorially specifying genetic components

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Subu K Subramanian ◽  
William P Russ ◽  
Rama Ranganathan
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
Dna Sequences ◽  
Gene Synthesis ◽  
Modular Architecture ◽  
Design And Synthesis ◽  
Genetic Components ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Primers ◽  
Microarray Chips

Abstract The design and synthesis of novel genes and deoxyribonucleic acid (DNA) sequences is a central technique in synthetic biology. Current methods of high throughput gene synthesis use pooled oligonucleotides obtained from custom-designed DNA microarray chips, and rely on orthogonal (non-interacting) polymerase chain reaction primers to specifically de-multiplex, by amplification, the precise subset of oligonucleotides necessary to assemble a full length gene. The availability of a large validated set of mutually orthogonal primers is therefore a crucial reagent for high-throughput gene synthesis. Here, we present a set of 166 20-nucleotide primers that are experimentally verified to be non-interacting, capable of specifying 13 695 unique genes. These primers represent a valuable resource to the synthetic biology community for specifying genetic components that can be assembled through a scalable and modular architecture.

Myriad post-Myriad

2020 ◽  
Author(s):  
Shubha Ghosh
Keyword(s):  
Dna Sequences ◽  
Negative Impact ◽  
Genetic Data ◽  
Brca1 And Brca2 ◽  
Minimal Impact ◽  
Synthetic Dna ◽  
The Us ◽  
Myriad Genetics ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Primers

Abstract The US Supreme Court’s 2013 decision, holding patent claims to isolated, endogenous deoxyribonucleic acid (DNA) sequences to be invalid, seemed to have limited negative impact on Myriad Genetics whose patent on the isolated BRCA1 and BRCA2 genes were at the heart of the case. This article explains this minimal impact in two ways. First, the Court’s decision still left synthetic DNA patentable, leaving that as a fruitful source for commercialization by companies like Myriad. The Federal Circuit’s subsequent decision, however, invalidated Myriad’s product claims over the synthetic polymerase chain reaction primers based on the isolated DNA sequences. Second, the Court’s decision did not address the patentability of mined genetic data for diagnostic and therapeutic purposes. This field of genetic data mining is precisely where Myriad has moved in its patenting activity.

scholarly journals Standardizing Automated DNA Assembly: Best Practices, Metrics, and Protocols Using Robots

2019 ◽  
Vol 24 (3) ◽  
pp. 282-290 ◽  
Author(s):  
David I. Walsh ◽  
Marilene Pavan ◽  
Luis Ortiz ◽  
Scott Wick ◽  
Johanna Bobrow ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Best Practices ◽  
High Throughput ◽  
Dna Sequences ◽  
Biological Systems ◽  
Software Tool ◽  
Parameter Analysis ◽  
Dna Assembly ◽  
Assembly Design ◽  
Liquid Handling

The advancement of synthetic biology requires the ability to create new DNA sequences to produce unique behaviors in biological systems. Automation is increasingly employed to carry out well-established assembly methods of DNA fragments in a multiplexed, high-throughput fashion, allowing many different configurations to be tested simultaneously. However, metrics are required to determine when automation is warranted based on factors such as assembly methodology, protocol details, and number of samples. The goal of our synthetic biology automation work is to develop and test protocols, hardware, and software to investigate and optimize DNA assembly through quantifiable metrics. We performed a parameter analysis of DNA assembly to develop a standardized, highly efficient, and reproducible MoClo protocol, suitable to be used both manually and with liquid-handling robots. We created a key DNA assembly metric (Q-metric) to characterize a given automation method’s advantages over conventional manual manipulations with regard to researchers’ highest-priority parameters: output, cost, and time. A software tool called Puppeteer was developed to formally capture these metrics, help define the assembly design, and provide human and robotic liquid-handling instructions. Altogether, we contribute to a growing foundation of standardizing practices, metrics, and protocols for automating DNA assembly.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

Using a Polymerase Chain Reaction as a Complementary Test to Improve the Detection of Dermatophyte Fungus in Nails

2014 ◽  
Vol 104 (3) ◽  
pp. 233-237 ◽  
Author(s):  
María José Iglesias Sánchez ◽  
Ana María Pérez Pico ◽  
Félix Marcos Tejedor ◽  
María Jesús Iglesias Sánchez ◽  
Raquel Mayordomo Acevedo
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Classical Method ◽  
Clinical Manifestations ◽  
Culture Method ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Traditional Procedure ◽  
Polymerase Chain

Background Dermatomycoses are a group of pathologic abnormalities frequently seen in clinical practice, and their prevalence has increased in recent decades. Diagnostic confirmation of mycotic infection in nails is essential because there are several pathologic conditions with similar clinical manifestations. The classical method for confirming the presence of fungus in nail is microbiological culture and the identification of morphological structures by microscopy. Methods We devised a nested polymerase chain reaction (PCR) that amplifies specific DNA sequences of dermatophyte fungus that is notably faster than the 3 to 4 weeks that the traditional procedure takes. We compared this new technique and the conventional plate culture method in 225 nail samples. The results were subjected to statistical analysis. Results We found concordance in 78.2% of the samples analyzed by the two methods and increased sensitivity when simultaneously using the two methods to analyze clinical samples. Now we can confirm the presence of dermatophyte fungus in most of the positive samples in just 24 hours, and we have to wait for the result of culture only in negative PCR cases. Conclusions Although this PCR cannot, at present, substitute for the traditional culture method in the detection of dermatophyte infection of the nails, it can be used as a complementary technique because its main advantage lies in the significant reduction of time used for diagnosis, in addition to higher sensitivity.

scholarly journals A Synthetic Biology Approach Using Engineered Bacteria to Detect Perfluoroalkyl Substance (PFAS) Contamination in Water

2021 ◽  
Vol 186 (Supplement_1) ◽  
pp. 801-807
Author(s):  
Nathaniel A Young ◽  
Ryan L Lambert ◽  
Angela M Buch ◽  
Christen L Dahl ◽  
Jackson D Harris ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequences ◽  
Detection System ◽  
The United States ◽  
Detection Methods ◽  
Health Concern ◽  
Contaminated Water ◽  
Engineered Bacteria ◽  
Synthetic Biology Approach ◽  
Rhodococcus Jostii

ABSTRACT Introduction Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds used industrially for a wide variety of applications. These PFAS compounds are very stable and persist in the environment. The PFAS contamination is a growing health issue as these compounds have been reported to impact human health and have been detected in both domestic and global water sources. Contaminated water found on military bases poses a potentially serious health concern for active duty military, their families, and the surrounding communities. Previous detection methods for PFAS in contaminated water samples require expensive and time-consuming testing protocols that limit the ability to detect this important global pollutant. The main objective of this work was to develop a novel detection system that utilizes a biological reporter and engineered bacteria as a way to rapidly and efficiently detect PFAS contamination. Materials and Methods The United States Air Force Academy International Genetically Engineered Machine team is genetically engineering Rhodococcus jostii strain RHA1 to contain novel DNA sequences composed of a propane 2-monooxygenase alpha (prmA) promoter and monomeric red fluorescent protein (mRFP). The prmA promoter is activated in the presence of PFAS and transcribes the mRFP reporter. Results The recombinant R. jostii containing the prmA promoter and mRFP reporter respond to exposure of PFAS by activating gene expression of the mRFP. At 100 µM of perfluorooctanoic acid, the mRFP expression was increased 3-fold (qRT-PCR). Rhodococcus jostii without exposure to PFAS compounds had no mRFP expression. Conclusions This novel detection system represents a synthetic biology approach to more efficiently detect PFAS in contaminated samples. With further refinement and modifications, a similar system could be readily deployed in the field around the world to detect this critical pollutant.

Sign in / Sign up

Export Citation Format

Share Document