scholarly journals A Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools

2021 ◽  
Author(s):  
Claris Winston ◽  
Lee Organick ◽  
Luis Ceze ◽  
Karin Strauss ◽  
Yuan-Jyue Chen
Keyword(s):  
Data Storage ◽  
Large Scale ◽  
High Specificity ◽  
Pcr Primers ◽  
Dna Database ◽  
Dna Storage ◽  
Wet Lab ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Address System

ABSTRACTWith the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications, including gene synthesis, CRISPR-based gene editing, and DNA data storage. Selectively retrieving specific oligos from these complex pools traditionally uses Polymerase Chain Reaction (PCR), in which any selected oligos are exponentially amplified to quickly outnumber non-selected ones. In this case, the number of orthogonal PCR primers is limited due to interactions between them. This lack of specificity presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool (i.e., a DNA database) is orders of magnitude larger than it is for other applications. Although a nested file address system was recently developed to increase the number of accessible files for DNA storage, it requires a more complicated lab protocol and more expensive reagents to achieve high specificity. Instead, we developed a new combinatorial PCR method that outperforms prior work without compromising the fidelity of retrieved material or complicating wet lab processes. Our method quadratically increases the number of accessible oligos while maintaining high specificity. In experiments, we accessed three arbitrarily chosen files from a DNA prototype database that contained 81 different files. Initially comprising only 1% of the original database, the selected files were enriched to over 99.9% using our combinatorial primer method. Our method thus provides a viable path for scaling up DNA data storage systems and has broader utility whenever scientists need access to a specific target oligo and can design their own primer regions.

scholarly journals Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse-Transcription PCR

Plant Disease ◽  
2007 ◽  
Vol 91 (11) ◽  
pp. 1496-1501 ◽  
Author(s):  
P. Margaria ◽  
C. Rosa ◽  
C. Marzachì ◽  
M. Turina ◽  
S. Palmano
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Diagnostic Methods ◽  
Kappa Index ◽  
Rt Pcr ◽  
Flavescence Dorée ◽  
Reverse Transcription Pcr ◽  
Novel Method ◽  
Pcr Method ◽  
Polymerase Chain

Flavescence dorée (FD) is the most serious phytoplasma disease of grapevine. This report describes a novel method of detecting FD phytoplasma based on reverse-transcription polymerase chain reaction (RT-PCR) on 16S ribosomal RNA (16SrRNA) which will greatly improve mass screening of infected grapevines. A rapid protocol for extracting sap from whole leaves or midveins and successive one-tube amplification by RT-PCR was applied to grapevine samples with or without symptoms collected from different areas of Piedmont (northwestern Italy). Results were compared with those obtained using one of the current diagnostic methods that utilizes nested PCR on phytoplasma DNA-enriched preparations. A Cohen's kappa index of 0.76 indicated a substantial agreement between the two sets of results. The RT-PCR method has the advantage of being a rapid, reliable, and sensitive assay for large-scale screening of grapevines.

Identification of colonization factors of enterotoxigenicEscherichia coliwith PCR-based technique

2009 ◽  
Vol 138 (4) ◽  
pp. 519-524 ◽  
Author(s):  
O. PUIPROM ◽  
S. CHANTAROJ ◽  
W. GANGNONNGIW ◽  
K. OKADA ◽  
T. HONDA ◽  
...  
Keyword(s):  
Protective Immunity ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Colonization Factor ◽  
Pcr Method ◽  
Colonization Factors ◽  
Polymerase Chain ◽  
Colonization Factor Antigen I ◽  
Factor Antigen

SUMMARYColonization factors (CFs) mediate attachment of enterotoxigenicEscherichia coli(ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhoea. We designed CF-specific polymerase chain reaction (PCR) primers, and developed a simple PCR-based genotypic CF identification method. ETEC strains (n=17) isolated from patients with diarrhoea in Thailand were examined for genotypical identification of CFs of ETEC strains. Coli surface antigen 6 (CS6) was the most common CF (29%), followed by CS13 (12%), colonization factor antigen I (CFA/I), CS2 and CS3, and CS17/CS19 (6% each), while 41% of the strains were negative. This simple PCR method for the detection of CF genes is useful for surveillance of ETEC infections in diagnostic laboratories.

scholarly journals Driving the scalability of DNA-based information storage systems

2019 ◽  
Author(s):  
Kyle J. Tomek ◽  
Kevin Volkel ◽  
Alexander Simpson ◽  
Austin G. Hass ◽  
Elaine W. Indermaur ◽  
...  
Keyword(s):  
Data Storage ◽  
Storage Systems ◽  
Information Storage ◽  
Maximum Capacity ◽  
Theoretical Maximum ◽  
New Approaches ◽  
File Access ◽  
Storage Media ◽  
Dna Storage ◽  
Address System

ABSTRACTThe extreme density of DNA presents a compelling advantage over current storage media; however, in order to reach practical capacities, new approaches for organizing and accessing information are needed. Here we use chemical handles to selectively extract unique files from a complex database of DNA mimicking 5 TB of data and design and implement a nested file address system that increases the theoretical maximum capacity of DNA storage systems by five orders of magnitude. These advancements enable the development and future scaling of DNA-based data storage systems with reasonable modern capacities and file access capabilities.

scholarly journals Analysis and Forecasting of Global RT-PCR Primers for SARS-CoV-2

2020 ◽  
Author(s):  
Gowri Nayar ◽  
Ed Seabolt ◽  
Mark Kunitomi ◽  
Akshay Agarwal ◽  
Kristen L. Beck ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Animal Cell ◽  
Geographic Location ◽  
High Specificity ◽  
Pcr Primers ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Over Time

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However, the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

scholarly journals Application of PCR-ELISA in Molecular Diagnosis

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Mei Jean Sue ◽  
Swee Keong Yeap ◽  
Abdul Rahman Omar ◽  
Sheau Wei Tan
Keyword(s):  
Detection Limit ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Pcr Product ◽  
Diagnostic Time ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Agricultural Industries ◽  
Analytical Time

Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality.

scholarly journals Content-Based Similarity Search in Large-Scale DNA Data Storage Systems

2020 ◽  
Author(s):  
Callista Bee ◽  
Yuan-Jyue Chen ◽  
David Ward ◽  
Xiaomeng Liu ◽  
Georg Seelig ◽  
...  
Keyword(s):  
Data Storage ◽  
Dna Sequences ◽  
Similarity Search ◽  
Large Scale ◽  
Storage Systems ◽  
Unique Identifier ◽  
Query Image ◽  
Synthetic Dna ◽  
Dna Database ◽  
Similar Images

AbstractSynthetic DNA has the potential to store the world’s continuously growing amount of data in an extremely dense and durable medium. Current proposals for DNA-based digital storage systems include the ability to retrieve individual files by their unique identifier, but not by their content. Here, we demonstrate content-based retrieval from a DNA database by learning a mapping from images to DNA sequences such that an encoded query image will retrieve visually similar images from the database via DNA hybridization. We encoded and synthesized a database of 1.6 million images and queried it with a variety of images, showing that each query retrieves a sample of the database containing visually similar images are retrieved at a rate much greater than chance. We compare our results with several algorithms for similarity search in electronic systems, and demonstrate that our molecular approach is competitive with state-of-the-art electronics.One Sentence SummaryLearned encodings enable content-based image similarity search from a database of 1.6 million images encoded in synthetic DNA.

scholarly journals A Novel Multiplex Polymerase Chain Reaction Approach for Detection of Four Human Infective Cryptosporidium Isolates: Cryptosporidium Parvum, types H and C, Cryptosporidium Canis, and Cryptosporidium Felis in Fecal and Soil Samples

2003 ◽  
Vol 15 (3) ◽  
pp. 262-267 ◽  
Author(s):  
Gabriella Lindergard ◽  
Daryl V. Nydam ◽  
Susan E. Wade ◽  
Stephanie L. Schaaf ◽  
Hussni O. Mohammed
Keyword(s):  
Polymerase Chain Reaction ◽  
Cryptosporidium Parvum ◽  
Multiplex Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
High Specificity ◽  
Pcr Primers ◽  
Cross Reactivity ◽  
Chain Reaction ◽  
Polymerase Chain

A nested multiplex polymerase chain reaction (PCR) approach was adopted for the simultaneous detection of 4 human infective genotypes of the protozoan parasite Cryptosporidium. Specific PCR primers were designed for the heat shock protein 70 gene of 2 genotypes of Cryptosporidium parvum (human and bovine types), Cryptosporidium canis, and Cryptosporidium felis. These 4 genotypes have all been found in human fecal samples. The primers amplified DNA fragments of specific sizes, each representing a unique genotype. The limit of detection of the method was found to vary between 10 and 100 oocysts per 1 ml fecal material. There appeared to be no cross-reactivity with other organisms commonly present in feces and soil, and the approach has a high specificity. The rapid identification of various human infective Cryptosporidium isolates is a part of the authors' long-term aim of determining the routes of infection with oocysts and thereby increase their epidemiological understanding of Cryptosporidium infection in humans and animals.

scholarly journals Multidimensional Data Organization and Random Access in Large-Scale DNA Storage Systems

2019 ◽  
Author(s):  
Xin Song ◽  
Shalin Shah ◽  
John Reif
Keyword(s):  
Large Scale ◽  
Storage Systems ◽  
Random Access ◽  
Data Retrieval ◽  
Pcr Primers ◽  
Multidimensional Data ◽  
Data Organization ◽  
Dna Storage ◽  
Reverse Primer ◽  
Access Patterns

AbstractWith impressive density and coding capacity, DNA offers a promising solution for building long-lasting data archival storage systems. In recent implementations, data retrieval such as random access typically relies on a large library of non-interacting PCR primers. While several algorithms automate the primer design process, the capacity and scalability of DNA-based storage systems are still fundamentally limited by the availability of experimentally validated orthogonal primers. In this work, we combine the nested and semi-nested PCR techniques to virtually enforce multidimensional data organization in large DNA storage systems. The strategy effectively pushes the limit of DNA storage capacity and reduces the number of primers needed for efficient random access from very large address space. Specifically, our design requires k * n unique primers to index nk data entries, where k specifies the number of dimensions and n indicates the number of data entries stored in each dimension. We strategically leverage forward/reverse primer pairs from the same or different address layers to virtually specify and maintain data retrievals in the form of rows, columns, tables, and blocks with respect to the original storage pool. This architecture enables various random-access patterns that could be tailored to preserve the underlying data structures and relations (e.g., files and folders) within the storage content. With just one or two rounds of PCR, specific data subsets or individual datum from the large multidimensional storage can be selectively enriched for simple extraction by gel electrophoresis or readout via sequencing.Abstract Figure

scholarly journals Analysis and forecasting of global real time RT-PCR primers and probes for SARS-CoV-2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gowri Nayar ◽  
Edward E. Seabolt ◽  
Mark Kunitomi ◽  
Akshay Agarwal ◽  
Kristen L. Beck ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Animal Cell ◽  
Geographic Location ◽  
High Specificity ◽  
Pcr Primers ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Over Time

AbstractRapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

scholarly journals Analysis and Forecasting of Global RT-PCR Primers for SARS-CoV-2

2021 ◽  
Author(s):  
Gowri Nayar ◽  
Edward Seabolt ◽  
Mark Kunitomi ◽  
Akshay Agarwal ◽  
Kristen Beck ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Animal Cell ◽  
Geographic Location ◽  
High Specificity ◽  
Pcr Primers ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Over Time

Abstract Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

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