-
Molekuliarnaia Biologiia 2016"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful... (Review)
Review
"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.
Topics: Cloning, Molecular; DNA, Recombinant; Genetic Engineering; Genetic Vectors
PubMed: 27028806
DOI: 10.7868/S0026898416010134 -
Journal of Biosciences Jun 2019Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the... (Review)
Review
Restriction enzymes have been identified in the early 1950s of the past century and have quickly become key players in the molecular biology of DNA. Forty years ago, the scientists whose pioneering work had explored the activity and sequence specificity of these enzymes, contributing to the definition of their enormous potential as tools for DNA characterization, mapping and manipulation, were awarded the Nobel Prize. In this short review, we celebrate the history of these enzymes in the light of their many different uses, as these proteins have accompanied the history of DNA for over 50 years representing active witnesses of major steps in the field.
Topics: CRISPR-Cas Systems; Chromatin; Chromosome Mapping; Cloning, Molecular; DNA; DNA Methylation; DNA Restriction Enzymes; History, 20th Century; History, 21st Century; Humans; Molecular Biology; Nobel Prize; Nucleotide Mapping; Transcription Activator-Like Effector Nucleases
PubMed: 31180051
DOI: No ID Found -
Current Protocols Jul 2021The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a...
The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.
Topics: Animals; Chromatin; Chromosomes; Genome; High-Throughput Nucleotide Sequencing; Nucleic Acid Conformation
PubMed: 34286910
DOI: 10.1002/cpz1.198 -
Methods in Molecular Biology (Clifton,... 2015RFLP (Restriction Fragment Length Polymorphism) is a commonly used technique that can be used for genotyping for nearly all organisms, including plants, animals, and...
RFLP (Restriction Fragment Length Polymorphism) is a commonly used technique that can be used for genotyping for nearly all organisms, including plants, animals, and humans. RFLP is widely used in genetic and genomic research, such as genome mapping and gene identification. The technique involves DNA digestion, gel electrophoresis, capillary transfer of DNA, and southern hybridization. In this chapter, we aim to give a detailed introduction of how to perform RFLPs for identifying genotypes.
Topics: Blotting, Southern; DNA Probes; DNA, Plant; Genotyping Techniques; Membranes, Artificial; Nucleic Acid Hybridization; Polymorphism, Restriction Fragment Length; Radioisotopes
PubMed: 25373751
DOI: 10.1007/978-1-4939-1966-6_7 -
Annual Review of Immunology Apr 2022Vertebrate immune systems suppress viral infection using both innate restriction factors and adaptive immunity. Viruses mutate to escape these defenses, driving hosts to... (Review)
Review
Vertebrate immune systems suppress viral infection using both innate restriction factors and adaptive immunity. Viruses mutate to escape these defenses, driving hosts to counterevolve to regain fitness. This cycle recurs repeatedly, resulting in an evolutionary arms race whose outcome depends on the pace and likelihood of adaptation by host and viral genes. Although viruses evolve faster than their vertebrate hosts, their proteins are subject to numerous functional constraints that impact the probability of adaptation. These constraints are globally defined by evolutionary landscapes, which describe the fitness and adaptive potential of all possible mutations. We review deep mutational scanning experiments mapping the evolutionary landscapes of both host and viral proteins engaged in arms races. For restriction factors and some broadly neutralizing antibodies, landscapes favor the host, which may help to level the evolutionary playing field against rapidly evolving viruses. We discuss the biophysical underpinnings of these landscapes and their therapeutic implications.
Topics: Animals; Biological Evolution; Humans; Mutation; Viral Proteins; Virus Diseases; Viruses
PubMed: 35080919
DOI: 10.1146/annurev-immunol-072621-084422 -
Methods in Molecular Biology (Clifton,... 2017A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful...
A major goal for biologists is to understand the connection between genes and phenotypic traits, and genetic mapping in experimental populations remains a powerful approach for discovering the causal genes underlying phenotypes. For genetic mapping, the process of genotyping was previously a major rate-limiting step. Modern sequencing technology has greatly improved the resolution and speed of genetic mapping by reducing the time, labor, and cost per genotyping marker. In addition, the ability to perform genotyping-by-sequencing (GBS) has facilitated large-scale population genetic analyses by providing a simpler way to survey segregating genetic variation in natural populations. Here we present two protocols for GBS, using the Illumina platform, that can be applied to a wide range of genotyping projects in different species. The first protocol is for genotyping a subset of marker positions genome-wide using restriction digestion, and the second is for preparing inexpensive paired-end whole-genome libraries. We discuss the suitability of each approach for different genotyping applications and provide notes for adapting these protocols for use with a liquid-handling robot.
Topics: Electrophoresis, Agar Gel; Genotype; High-Throughput Nucleotide Sequencing
PubMed: 27822868
DOI: 10.1007/978-1-4939-6442-0_16 -
PloS One 2021In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a...
In genomics, optical mapping technology provides long-range contiguity information to improve genome sequence assemblies and detect structural variation. Originally a laborious manual process, Bionano Genomics platforms now offer high-throughput, automated optical mapping based on chips packed with nanochannels through which unwound DNA is guided and the fluorescent DNA backbone and specific restriction sites are recorded. Although the raw image data obtained is of high quality, the processing and assembly software accompanying the platforms is closed source and does not seem to make full use of data, labeling approximately half of the measured signals as unusable. Here we introduce two new software tools, independent of Bionano Genomics software, to extract and process molecules from raw images (OptiScan) and to perform molecule-to-molecule and molecule-to-reference alignments using a novel signal-based approach (OptiMap). We demonstrate that the molecules detected by OptiScan can yield better assemblies, and that the approach taken by OptiMap results in higher use of molecules from the raw data. These tools lay the foundation for a suite of open-source methods to process and analyze high-throughput optical mapping data. The Python implementations of the OptiTools are publicly available through http://www.bif.wur.nl/.
Topics: Chromosome Mapping; Genomics; High-Throughput Nucleotide Sequencing; Optical Restriction Mapping; Sequence Analysis, DNA
PubMed: 34591846
DOI: 10.1371/journal.pone.0253102 -
Methods in Molecular Biology (Clifton,... 2015Quantitative trait locus (QTL) mapping in plants dates to the 1980s (Stuber et al. Crop Sci 27: 639-648, 1987; Paterson et al. Nature 335: 721-726, 1988), but earlier... (Review)
Review
Quantitative trait locus (QTL) mapping in plants dates to the 1980s (Stuber et al. Crop Sci 27: 639-648, 1987; Paterson et al. Nature 335: 721-726, 1988), but earlier studies were often hindered by the expense and time required to identify large numbers of polymorphic genetic markers that differentiated the parental genotypes and then to genotype them on large segregating mapping populations. High-throughput sequencing has provided an efficient means to discover single nucleotide polymorphisms (SNPs) that can then be assayed rapidly on large populations with array-based techniques (Gupta et al. Heredity 101: 5-18, 2008). Alternatively, high-throughput sequencing methods such as restriction site-associated DNA sequencing (RAD-Seq) (Davey et al. Nat Rev Genet 12: 499-510, 2011; Baird et al. PloS ONE 3: e3376, 2008) and genotyping-by-sequencing (GBS) (Elshire et al. PLoS One 6: 2011; Glaubitz et al. PLoS One 9: e90346, 2014) can be used to identify and genotype polymorphic markers directly. Linkage disequilibrium (LD) between markers and causal variants is needed to detect QTL. The earliest QTL mapping methods used backcross and F2 generations of crosses between inbred lines, which have high levels of linkage disequilibrium (dependent entirely on the recombination frequency between chromosomal positions), to ensure that QTL would have sufficiently high linkage disequilibrium with one or more markers on sparse genetic linkage maps. The downside of this approach is that resolution of QTL positions is poor. The sequencing technology revolution, by facilitating genotyping of vastly more markers than was previously feasible, has allowed researchers to map QTL in situations of lower linkage disequilibrium, and consequently, at higher resolution. We provide a review of methods to identify QTL with higher precision than was previously possible. We discuss modifications of the traditional biparental mapping population that provide higher resolution of QTL positions, QTL fine-mapping procedures, and genome-wide association studies, all of which are greatly facilitated by high-throughput sequencing methods. Each of these procedures has many variants, and consequently many details to consider; we focus our chapter on the consequences of practical decisions that researchers make when designing QTL mapping studies and when analyzing the resulting data. The ultimate goal of many of these studies is to resolve a QTL to its causal sequence variation.
Topics: Chromosome Mapping; Cloning, Molecular; Crosses, Genetic; Genetic Markers; Genome-Wide Association Study; Genotyping Techniques; High-Throughput Nucleotide Sequencing; Linkage Disequilibrium; Phenotype; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Quantitative Trait Loci
PubMed: 25757777
DOI: 10.1007/978-1-4939-2444-8_13 -
GigaScience 2015Optical Mapping is an established single-molecule, whole-genome analysis system, which has been used to gain a comprehensive understanding of genomic structure and to... (Review)
Review
Optical Mapping is an established single-molecule, whole-genome analysis system, which has been used to gain a comprehensive understanding of genomic structure and to study structural variation of complex genomes. A critical component of Optical Mapping system is the image processing module, which extracts single molecule restriction maps from image datasets of immobilized, restriction digested and fluorescently stained large DNA molecules. In this review, we describe robust and efficient image processing techniques to process these massive datasets and extract accurate restriction maps in the presence of noise, ambiguity and confounding artifacts. We also highlight a few applications of the Optical Mapping system.
Topics: Animals; Chromosome Mapping; Genome; Humans; Image Processing, Computer-Assisted; Sensitivity and Specificity
PubMed: 26617984
DOI: 10.1186/s13742-015-0096-z -
GigaScience 2014Optical mapping and newer genome mapping technologies based on nicking enzymes provide low resolution but long-range genomic information. The optical mapping technique... (Review)
Review
Optical mapping and newer genome mapping technologies based on nicking enzymes provide low resolution but long-range genomic information. The optical mapping technique has been successfully used for assessing the quality of genome assemblies and for detecting large-scale structural variants and rearrangements that cannot be detected using current paired end sequencing protocols. Here, we review several algorithms and methods for building consensus optical maps and aligning restriction patterns to a reference map, as well as methods for using optical maps with sequence assemblies.
PubMed: 25671093
DOI: 10.1186/2047-217X-3-33