Overcoming variant mutation-related impacts on viral sequencing and detection methodologies.

Prompt and accurate pathogen identification, by diagnostics and sequencing, is an effective tool for tracking and potentially curbing pathogen spread. Targeted detection and amplification of viral genomes depends on annealing complementary oligonucleotides to genomic DNA or cDNA. However, genomic mutations that occur during viral evolution may perturb annealing, which can result in incomplete sequence coverage of the genome and/or false negative diagnostic test results. Herein, we demonstrate how to assess, test, and optimize sequencing and detection methodologies to attenuate the negative impact of mutations on genome targeting efficiency. This evaluation was conducted using in vitro-transcribed (IVT) RNA as well as RNA extracted from clinical SARS-CoV-2 variant samples, including the heavily mutated Omicron variant. Using SARS-CoV-2 as a current example, these results demonstrate how to maintain reliable targeted pathogen sequencing and how to evaluate detection methodologies as new variants emerge.

E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity.

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.

Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP).

Human spaceflight endeavors present an opportunity to expand our presence beyond Earth. To this end, it is crucial to understand and diagnose effects of long-term space travel on the human body. Developing tools for targeted, on-site detection of specific DNA sequences will allow us to establish research and diagnostics platforms that will benefit space programs. We describe a simple DNA diagnostic method that utilizes colorimetric loop-mediated isothermal amplification (LAMP) to enable detection of a repetitive telomeric DNA sequence in as little as 30 minutes. A proof of concept assay for this method was carried out using existing hardware on the International Space Station and the results were read instantly by an astronaut through a simple color change of the reaction mixture. LAMP offers a novel platform for on-orbit DNA-based diagnostics that can be deployed on the International Space Station and to the broader benefit of space programs.

Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other.

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

Erratum: Author Correction: Successful amplification of DNA aboard the International Space Station.

[This corrects the article DOI: 10.1038/s41526-017-0033-9.].

Successful amplification of DNA aboard the International Space Station.

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions.

Database of DNA polymerases.

The DNA Polymerase Database (Polbase) is intended to compile the wealth of existing DNA polymerase information from public and private records into an open, searchable database.

Polbase: a repository of biochemical, genetic and structural information about DNA polymerases.

Polbase (http://polbase.neb.com) is a freely accessible database of DNA polymerases and related references. It has been developed in a collaborative model with experts whose contributions reflect their varied backgrounds in genetics, structural biology and biochemistry. Polbase is designed to compile detailed results of polymerase experimentation, presenting them in a dynamic view to inform further research. After validation, results from references are displayed in context with relevant experimental details and are always traceable to their source publication. Polbase is connected to other resources, including PubMed, UniProt and the RCSB Protein Data Bank, to provide multi-faceted views of polymerase knowledge. In addition to a simple web interface, Polbase data is exposed for custom analysis by external software. With the contributions of many polymerase investigators, Polbase has become a powerful research tool covering most important aspects of polymerases, from sequence and structure to biochemistry.

DNA ligases.

The DNA ligase enzyme family catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA. This activity can seal nicks in duplex DNA or join double-stranded DNA fragments having either blunt or cohesive ends. DNA ligases are central enzymes in molecular biology, nucleic acid research, and in next-generation sequencing applications. Reaction conditions and applications for T4 DNA ligase, E. coli DNA ligase, and thermostable DNA ligases are described in this unit. These enzymes differ in their cofactor requirements, substrate specificity, and thermal stability.

Endonucleases.

Reaction conditions for a variety of endonucleases are detailed in this unit along with discussions of potential applications. Enzymes covered include BAL 31 nuclease, S1 nuclease, mung bean nuclease, micrococcal nuclease, and DNase I. A general discussion regarding the use of endonucleases to generate nonspecific breaks in dsDNA is also provided. For a detailed discussion of the endonucleases more typically associated with DNA damage repair (e.g., Endo III, IV, V and VIII of E. coli and human APE1), see UNIT 3.9.

DNA cloning and engineering by uracil excision.

This unit describes a simple and efficient DNA engineering method that combines nucleotide sequence alteration, multiple PCR fragment assembly, and directional cloning. PCR primers contain a single deoxyuracil residue (dU), and can be designed to accommodate nucleotide substitutions, insertions, and/or deletions. The primers are then used to amplify DNA in discrete fragments that incorporate a dU at each end. Excision of deoxyuracils results in PCR fragments flanked by unique, overlapping, single-stranded extensions that allow the seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment. Two basic protocols on the methods of uracil excision-based engineering are presented, and special attention is given to primer design. The use of a commercially available cloning vector and the preparation of custom vectors are also presented.

Ribonucleases.

Ribonucleases (RNases) with different sequence or structural specificities are used for a variety of analytical purposes, including RNA sequencing, mapping, and quantitation. The development of RNase protection assays, structural determination assays, and the production of small interfering RNAs (siRNA) employed in RNA interference (RNAi) experiments has depended on the unique substrate specificities of commercially available RNases, including RNases A, I, T1, V1, HI, III, and Dicer. One very common application for high purity RNase A is also presented in this unit and involves hydrolyzing RNA that contaminates DNA preparations. RNase HII and the placental RNase inhibitor are also discussed.

RNA ligases.

T4 RNA ligase 1 catalyzes the ATP-dependent covalent joining of single-stranded 5'-phosphoryl termini of DNA or RNA to single-stranded 3'-hydroxyl termini of DNA or RNA. T4 RNA ligase 2 also catalyzes the joining of a 3'-hydroxyl terminus of RNA to a 5'-phosphorylated RNA or DNA; unlike T4 RNA ligase 1, this enzyme prefers double-stranded substrates. A truncated form of T4 RNA ligase 2 requires a pre-adenylated substrate for ligation. This unit describes specific reaction conditions, as well as applications such as radioactive labeling of the 3' termini of RNA, circularizing oligodeoxyribonucleotides and oligoribonucleotides, ligating oligomers and nicks, creating hybrid and chimeric DNA/RNA molecules, and miRNA cloning.

DNA-dependent DNA polymerases.

This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion-bypass DNA polymerases that play a role in DNA damage repair.

Template-independent DNA polymerases.

Terminal deoxynucleotidyl transferase (TdT), is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides at the 3'-hydroxyl terminus of DNA, accompanied by the release of inorganic phosphate. TdT does not require a template and will not copy one. Reaction conditions and some applications are described in this unit, including cloning DNA fragments, labeling the 3' terminus of DNA with (32)P or nonradioactive tags, synthesizing model polydeoxynucleotide homopolymers, and detecting DNA damage and apoptotic cells.

RNA-dependent DNA polymerases.

Reverse transcriptases (RTs) are multifunctional enzymes, but are mainly used as RNA-directed DNA polymerases in first-strand cDNA synthesis. Specifically, oligodeoxynucleotides are used as primers for extension on RNA templates. The DNA synthesized from an RNA template is referred to as complementary DNA (cDNA) and is often used as a template for PCR or converted to dsDNA for cloning. This unit describes appropriate reaction conditions for RTs from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV), along with applications such as synthesizing cDNA, 3' fill-in reactions, and labeling the 3' terminus of DNA fragments with 5' protruding ends, and DNA sequencing.

DNA repair enzymes.

In vivo DNA damage impacts the genetic stability of an organism; therefore, multiple pathways utilizing a large number of enzymes have evolved to repair DNA damage. This unit focuses on enzymes involved in base excision repair (BER). The BER enzymes possessing N-glycosylase activity can find and remove a wide variety of damaged bases in a sea of normal bases. The combination of unique substrate specificity, accuracy, and robust in vitro activity of many of these enzymes has led to their use in various experimental techniques, including site-specific DNA cleavage. The enzymes described in this unit are active on many substrates including oxidized purines and pyrimidines, alkylated bases, abasic sites, pyrimidine dimers, deaminated cytosines, and deaminated adenines.

RNA polymerases.

This unit describes DNA-dependent, RNA-dependent, and template-independent RNA polymerases. DNA-dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA-dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template-independent polymerase, catalyzes the incorporation of AMP residues onto the free 3'-hydroxyl terminus of RNA, utilizing ATP as a precursor. Specific reaction conditions of poly(A) polymerase, as well as applications including RNA tailing and 3' end labeling, are discussed.