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Methods in Molecular Biology (Clifton,... 2023The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the...
The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.
Topics: Cloning, Molecular; DNA Ligase ATP; DNA Ligases; DNA Primers; Nucleotidyltransferases
PubMed: 36853455
DOI: 10.1007/978-1-0716-3004-4_4 -
Methods in Molecular Biology (Clifton,... 2023Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a...
Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2 kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.
Topics: DNA, Recombinant; Escherichia coli; Cloning, Molecular; Polymerase Chain Reaction; Genetic Vectors; DNA Ligase ATP
PubMed: 36853452
DOI: 10.1007/978-1-0716-3004-4_1 -
Cold Spring Harbor Protocols Feb 2019The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as...
The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences.
Topics: DNA; DNA Ligases; DNA Primers; DNA Restriction Enzymes; DNA, Circular; Polymerase Chain Reaction
PubMed: 30710023
DOI: 10.1101/pdb.prot095166 -
Current Opinion in Structural Biology Dec 2018Bacterial replisomes are dynamic multiprotein DNA replication machines that are inherently difficult for structural studies. However, breakthroughs continue to come. The... (Review)
Review
Bacterial replisomes are dynamic multiprotein DNA replication machines that are inherently difficult for structural studies. However, breakthroughs continue to come. The structures of Escherichia coli DNA polymerase III (core)-clamp-DNA subcomplexes solved by single-particle cryo-electron microscopy in both polymerization and proofreading modes and the discovery of the stochastic nature of the bacterial replisomes represent notable progress. The structures reveal an intricate interaction network in the polymerase-clamp subassembly, providing insights on how replisomes may work. Meantime, ensemble and single-molecule functional assays and fluorescence microscopy show that the bacterial replisomes can work in a decoupled and uncoordinated way, with polymerases quickly exchanging and both leading-strand and lagging-strand polymerases and the helicase working independently, contradictory to the elegant textbook view of a highly coordinated machine.
Topics: Bacillus subtilis; Bacteriophage T7; DNA Helicases; DNA Ligases; DNA Polymerase I; DNA Polymerase III; DNA Replication; DNA-Directed DNA Polymerase; Escherichia coli; Helicobacter pylori; Multienzyme Complexes
PubMed: 30292863
DOI: 10.1016/j.sbi.2018.09.006 -
Nucleic Acids Research Jun 2017DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly...
DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
Topics: Animals; Bone and Bones; DNA; DNA Ligases; DNA Primers; DNA, Single-Stranded; Fossils; Gene Library; High-Throughput Nucleotide Sequencing; Horses; Humans; Liver; Nucleic Acid Hybridization; Oligonucleotides; Polymerase Chain Reaction; Sequence Analysis, DNA; Swine
PubMed: 28119419
DOI: 10.1093/nar/gkx033 -
Nature Biotechnology May 2015The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR)...
The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.
Topics: Adenoviridae; Adenovirus E4 Proteins; Animals; CRISPR-Cas Systems; Cell Line; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; Gene Expression Regulation; Genetic Engineering; Genome, Human; Homologous Recombination; Humans; Mice; Viral Proteins
PubMed: 25803306
DOI: 10.1038/nbt.3198 -
Cold Spring Harbor Protocols Aug 2019DNA ligases are used chiefly to create novel combinations of nucleic acid molecules and to attach them to vectors before molecular cloning. They are either of bacterial...
DNA ligases are used chiefly to create novel combinations of nucleic acid molecules and to attach them to vectors before molecular cloning. They are either of bacterial origin or bacteriophage encoded and have different properties, as discussed here.
Topics: Bacteriophage T4; DNA; DNA Ligases; Enzyme Stability; Escherichia coli; RNA; Temperature
PubMed: 31371476
DOI: 10.1101/pdb.top101352 -
DNA Repair Sep 2020To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost... (Review)
Review
To ensure genome integrity, the joining of breaks in the phosphodiester backbone of duplex DNA is required during DNA replication and to complete the repair of almost all types of DNA damage. In human cells, this task is accomplished by DNA ligases encoded by three genes, LIG1, LIG3 and LIG4. Mutations in LIG1 and LIG4 have been identified as the causative factor in two inherited immunodeficiency syndromes. Moreover, there is emerging evidence that DNA ligases may be good targets for the development of novel anti-cancer agents. In this graphical review, we provide an overview of the roles of the DNA ligases encoded by the three human LIG genes in DNA replication and repair.
Topics: DNA; DNA Damage; DNA Ligase ATP; DNA Repair; DNA Replication; Humans; Poly-ADP-Ribose Binding Proteins
PubMed: 33087274
DOI: 10.1016/j.dnarep.2020.102908 -
Biochemical Society Transactions Jun 2016Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation,... (Review)
Review
Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved. This review highlights methods currently used for the synthesis of moderately long chemically modified nucleic acids (up to 1000 bp), their limitations and areas for future expansion.
Topics: Adenine; Cytosine; DNA; DNA Ligases; DNA-Directed DNA Polymerase; Guanine; Oligonucleotides; Polymerase Chain Reaction; Thymine
PubMed: 27284032
DOI: 10.1042/BST20160051 -
Frontiers in Microbiology 2023DNA ligase is an important enzyme ubiquitous in all three kingdoms of life that can ligate DNA strands, thus playing essential roles in DNA replication, repair and... (Review)
Review
DNA ligase is an important enzyme ubiquitous in all three kingdoms of life that can ligate DNA strands, thus playing essential roles in DNA replication, repair and recombination . , DNA ligase is also used in biotechnological applications requiring in DNA manipulation, including molecular cloning, mutation detection, DNA assembly, DNA sequencing, and other aspects. Thermophilic and thermostable enzymes from hyperthermophiles that thrive in the high-temperature (above 80°C) environments have provided an important pool of useful enzymes as biotechnological reagents. Similar to other organisms, each hyperthermophile harbors at least one DNA ligase. In this review, we summarize recent progress on structural and biochemical properties of thermostable DNA ligases from hyperthermophiles, focusing on similarities and differences between DNA ligases from hyperthermophilic bacteria and archaea, and between these thermostable DNA ligases and non-thermostable homologs. Additionally, altered thermostable DNA ligases are discussed. Possessing improved fidelity or thermostability compared to the wild-type enzymes, they could be potential DNA ligases for biotechnology in the future. Importantly, we also describe current applications of thermostable DNA ligases from hyperthermophiles in biotechnology.
PubMed: 37293226
DOI: 10.3389/fmicb.2023.1198784