Abstract:
:DNA assembly forms the cornerstone of modern synthetic biology. Despite the numerous available methods, scarless multi-fragment assembly of large plasmids remains challenging. Furthermore, the upcoming wave in molecular biological automation demands a rethinking of how we perform DNA assembly. To streamline automation workflow and minimize operator intervention, a non-enzymatic assembly method is highly desirable. Here, we report the optimization and operationalization of a process called Twin-Primer Assembly (TPA), which is a method to assemble polymerase chain reaction-amplified fragments into a plasmid without the use of enzymes. TPA is capable of assembling a 7 kb plasmid from 10 fragments at ∼80% fidelity and a 31 kb plasmid from five fragments at ∼50% fidelity. TPA cloning is scarless and sequence independent. Even without the use of enzymes, the performance of TPA is on par with some of the best in vitro assembly methods currently available. TPA should be an invaluable addition to a synthetic biologist's toolbox.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Liang J,Liu Z,Low XZ,Ang EL,Zhao Hdoi
10.1093/nar/gkx132subject
Has Abstractpub_date
2017-06-20 00:00:00pages
e94issue
11eissn
0305-1048issn
1362-4962pii
3045848journal_volume
45pub_type
杂志文章abstract::The Mouse Genome Database (MGD) (http://www.informatics.jax.org) one component of a community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology. MGD strives to provide an extensively integrated information resource with experimental d...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkg047
更新日期:2003-01-01 00:00:00
abstract::Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr890
更新日期:2012-02-01 00:00:00
abstract::Saccharomyces cerevisiae chl1 mutants have a significant increase in the rate of chromosome missegregation. CHL1 encodes a 99 kDa predicted protein with an ATP binding site consensus, a putative helix-turn-helix DNA binding motif, and homology to helicases. Using site-directed mutagenesis, I show that mutations that a...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/28.16.3056
更新日期:2000-08-15 00:00:00
abstract::Recent advances in next-generation sequencing technologies and genome assembly algorithms have enabled the accumulation of a huge volume of genome sequences from various species. This has provided new opportunities for large-scale comparative genomics studies. Identifying and utilizing synteny blocks, which are genomi...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw310
更新日期:2016-07-08 00:00:00
abstract::Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt1137
更新日期:2014-02-01 00:00:00
abstract::The STE2 gene of the yeast Saccharomyces cerevisiae encodes a component of the receptor for the oligopeptide pheromone alpha-factor. We have cloned and determined the nucleotide sequence of the STE2 gene. A sequence involved in the control of cell-type expression of the STE2 gene was found 5' of an open reading frame ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/13.23.8463
更新日期:1985-12-09 00:00:00
abstract::The higher order structure of the functionally important 530 loop in Escherichia coli 16S rRNA was studied in mutants with single base changes at position 517, which significantly impair translational fidelity. The 530 loop has been proposed to interact with the EF-Tu-GTP-aatRNA ternary complex during decoding. The re...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.17.3563
更新日期:1995-09-11 00:00:00
abstract::The rat alpha- and bovine alpha s1-casein genes have been isolated and their 5' sequences determined. The rat alpha-, beta-, gamma- and bovine alpha s1-casein genes contain similar 5' exon arrangements in which the 5' noncoding, signal peptide and casein kinase phosphorylation sequences are each encoded by separate ex...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.4.1883
更新日期:1986-02-25 00:00:00
abstract::Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm622
更新日期:2008-01-01 00:00:00
abstract::PRMT5 is an arginine methyltransferase that accounts for the vast majority of the symmetric methylation in cells. PRMT5 exerts its function when complexed with MEP50/WDR77. This activity is often elevated in cancer cells and correlates with poor prognosis, making PRMT5 a therapeutic target. To investigate the PRMT5 si...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz200
更新日期:2019-06-04 00:00:00
abstract::High throughput sequencing (HTS) platforms generate unprecedented amounts of data that introduce challenges for processing and downstream analysis. While tools that report the 'best' mapping location of each read provide a fast way to process HTS data, they are not suitable for many types of downstream analysis such a...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku370
更新日期:2014-07-01 00:00:00
abstract::The Escherichia coli cyclic AMP receptor protein (CRP) is a global regulator that controls transcription initiation from more than 100 promoters by binding to a specific DNA sequence within cognate promoters. Many genes in the CRP regulon have been predicted simply based on the presence of DNA-binding sites within gen...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkh908
更新日期:2004-11-01 00:00:00
abstract::To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/30.2.569
更新日期:2002-01-15 00:00:00
abstract::The vertebrate genome is a mosaic of regions differing dramatically in their G + C content. Those regions with a high G + C content contain the expected number of CpG dinucleotides and we propose that following methylation these have been protected from deamination by the increased stability of the surrounding DNA dup...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.14.5869
更新日期:1984-07-25 00:00:00
abstract::Alterations in cancer genomes strongly influence clinical responses to treatment and in many instances are potent biomarkers for response to drugs. The Genomics of Drug Sensitivity in Cancer (GDSC) database (www.cancerRxgene.org) is the largest public resource for information on drug sensitivity in cancer cells and mo...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gks1111
更新日期:2013-01-01 00:00:00
abstract::Recent studies have employed cross-species comparisons of transcription factor binding, reporting significant regulatory network 'rewiring' between species. Here, we address how a transcriptional repressor targets and regulates neural genes differentially between human and mouse embryonic stem cells (ESCs). We find th...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv514
更新日期:2015-07-13 00:00:00
abstract::A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/14.2.1073
更新日期:1986-01-24 00:00:00
abstract::Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and rep...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl115
更新日期:2006-04-14 00:00:00
abstract::MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky306
更新日期:2018-06-20 00:00:00
abstract::Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcrib...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.19.3795
更新日期:1997-10-01 00:00:00
abstract::Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleoso...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/9.17.4367
更新日期:1981-09-11 00:00:00
abstract::Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky162
更新日期:2018-05-18 00:00:00
abstract::Eukaryotic tRNAs are transcribed as precursors. A 5'-end leader and 3'-end trailer are endonucleolytically removed by RNase P and 3'-tRNase before 3'-end CCA addition, aminoacylation, nuclear export and translation. 3'-End -CC can be a 3'-tRNase anti-determinant with the ability to prevent mature tRNA from recycling t...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/29.21.4334
更新日期:2001-11-01 00:00:00
abstract::The webPDBinder (http://pdbinder.bio.uniroma2.it/PDBinder) is a web server for the identification of small ligand-binding sites in a protein structure. webPDBinder searches a protein structure against a library of known binding sites and a collection of control non-binding pockets. The number of similarities identifie...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt457
更新日期:2013-07-01 00:00:00
abstract::Mycobacterium tuberculosis (Mtb) Cmr (Rv1675c) is a CRP/FNR family transcription factor known to be responsive to cAMP levels and during macrophage infections. However, Cmr's DNA binding properties, cellular targets and overall role in tuberculosis (TB) complex bacteria have not been characterized. In this study, we u...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv889
更新日期:2016-01-08 00:00:00
abstract::A cross index is presented for using the improved selectivity offered by the Hung and Wensink (Nucl. Acids Res. 12, 1863-1874, 1984) method of partially filling in 5'-extensions produced by type II restriction endonucleases. After this treatment, DNA fragments which normally cannot be ligated to one another, can be jo...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/15.8.3199
更新日期:1987-04-24 00:00:00
abstract::Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, leading us to explore a connection between this pathway and the splicing machinery. We show here that addition of a r...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx213
更新日期:2017-06-20 00:00:00
abstract::In some preparations of DNA dependent RNA polymerase a new enzymatic activity has been found which catalyzes the condensation of two pyrophosphate molecules, liberated in the process of RNA synthesis, to one molecule of orthophosphate and one molecule of Mg (or Mn) - chelate complex with trimetaphosphate. This activit...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/6.4.1521
更新日期:1979-04-01 00:00:00
abstract::The cDNA of human DNA polymerase delta was cloned. The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa. Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb. Monoclonal and polyclonal antibodies to the C-terminal 20 residue...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/20.4.735
更新日期:1992-02-25 00:00:00
abstract::A coiled-coil microtubule-bundling protein, p180, was originally identified as one of the ribosome receptor candidates on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported that p180 plays crucial roles in upregulating collagen biosynthesis, mainly by facilitating ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr1197
更新日期:2012-04-01 00:00:00