Abstract：
:Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Chiu J,March PE,Lee R,Tillett Ddoi
10.1093/nar/gnh172keywords:
subject
Has Abstractpub_date
2004-12-07 00:00:00pages
e174issue
21eissn
0305-1048issn
1362-4962pii
32/21/e174journal_volume
32pub_type
杂志文章abstract：:The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal effi...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkq616
更新日期：2010-09-01 00:00:00
abstract：:Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGC...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/16.10.4607
更新日期：1988-05-25 00:00:00
abstract：:Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing a...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkaa826
更新日期：2020-11-04 00:00:00
abstract：:We have isolated a human cDNA (kup), encoding a new protein with two distantly spaced zinc fingers of the C2H2 type. This gene is highly conserved in mammals and is expressed mainly in hematopoietic cells and testis. Its expression was not higher in the various transformed cells tested than in the normal corresponding...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/19.7.1431
更新日期：1991-04-11 00:00:00
abstract：:We have examined the expression and structure of several genes belonging to two classes of vegetative specific genes of the simple eukaryote, Dictyostelium discoideum. In amebae grown on bacteria, deactivation of all vegetative specific genes occurred at the onset of development and very little mRNA exists by 8 to 10 ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/17.23.9679
更新日期：1989-12-11 00:00:00
abstract：:The beta protein encoded by the Streptococcus pyogenes plasmid pSM19035 is a site-specific recombinase involved in both resolution of plasmid multimers into monomers and DNA inversion. It has been proposed that the DNA region to which the beta recombinase binds to mediate recombination includes a promoter from which o...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/22.10.1855
更新日期：1994-05-25 00:00:00
abstract：:Addition of thiol compounds containing an anionic group to the 3'-terminal unsaturated sugar of the 5' fragment obtained from an oligonucleotide containing an AP site cleaved by beta-elimination, can be followed by gel electrophoresis. The technique enables to distinguish between two mechanisms of cleavage of the C3'-...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/16.20.9489
更新日期：1988-10-25 00:00:00
abstract：:We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The re...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/24.9.1710
更新日期：1996-05-01 00:00:00
abstract：:Actin is a major cytoskeletal protein in eukaryotes. Recent studies suggest more diverse functional roles for this protein. Actin mRNA is known to be localized to neuronal synapses and undergoes rapid deadenylation during early developmental stages. However, its 3'-untranslated region (UTR) is not characterized and th...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkn624
更新日期：2008-11-01 00:00:00
abstract：:The convergently transcribed restriction (R) and methylase (M) genes of the Restriction-Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vi...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkp210
更新日期：2009-06-01 00:00:00
abstract：:We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar struc...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkaa629
更新日期：2020-09-04 00:00:00
abstract：:RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkv752
更新日期：2015-09-18 00:00:00
abstract：:Unfolding of the nucleosomes in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3 and permits the selective recovery of the unfolded nucleosomes by mercury-affinity chromatography. This new technique has been used to compare the nucleosomal proteins and their postsynthetic modifications i...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/15.11.4375
更新日期：1987-06-11 00:00:00
abstract：:A refined map for the linear arrangement of histones along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5') H2B25,35-H455,65-H375,85,95/H488-H2B105,11 5-H2A118-H3135,145/H2A145 (3') (the subscripts giv...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/13.10.3439
更新日期：1985-05-24 00:00:00
abstract：:We describe a method for anchoring bacteriophage lambda DNA by one end to gold by Au-biotin-streptavidin-biotin-DNA bonds. DNA anchored to a microfabricated Au line could be aligned and stretched in flow and electric fields. The anchor was shown to resist a force of at least 11 pN, a linkage strong enough to allow DNA...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/22.3.492
更新日期：1994-02-11 00:00:00
abstract：:The vitamin D receptor mediates the signal of 1 alpha, 25-dihydroxyvitamin D3 by binding to vitamin D responsive elements in DNA as a homodimer or as a heterodimer composed of one vitamin D receptor subunit and one retinoid X receptor subunit. We have mapped the dimerization interfaces of the vitamin D receptor that i...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/23.4.606
更新日期：1995-02-25 00:00:00
abstract：:About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are e...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gnf051
更新日期：2002-06-15 00:00:00
abstract：:Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (approximately 10(-6) to 10(-8)), its practical application has been largely restricted to specific experimental systems that allow...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkp643
更新日期：2009-09-01 00:00:00
abstract：:An updated list of DNA sequences cloned from the human genome over the past year (1991) is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic probes comprising both gene and pseudogene sequences. ...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/20.suppl.2181
更新日期：1992-05-11 00:00:00
abstract：:Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/29.13.2757
更新日期：2001-07-01 00:00:00
abstract：:Eucaryotic transcription initiation by RNA polymerase II involves protein:DNA interactions during the formation of a transcription complex. In addition to RNA polymerase II there are at least five other general transcription factors necessary for initiation with the adenovirus major late promoter. One of these, TFIIA,...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/18.12.3611
更新日期：1990-06-25 00:00:00
abstract：:The nucleotide sequence for human argininosuccinate synthetase cDNA was determined by analysis of six clones isolated from a single experiment. The sequence covered 1623 nucleotides including 76 bases of poly(A) and contained a 1236 nucleotide open reading frame encoding a protein of 46,434 daltons. In one cDNA isolat...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/11.18.6505
更新日期：1983-09-24 00:00:00
abstract：:Dinucleoside diphosphates of the general type pGpN have been prepared enzymatically using ribonuclease N1. Alkylated uridines or cytidines, which are products of carcinogens acting on nucleic acids, were tested in dinucleoside diphosphates for their ability to stimulate the binding of Ala- or Val-tRNA to ribosomes. O2...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/6.4.1709
更新日期：1979-04-01 00:00:00
abstract：:This review presents detailed information about the structure of triplet repeat RNA and addresses the simple sequence repeats of normal and expanded lengths in the context of the physiological and pathogenic roles played in human cells. First, we discuss the occurrence and frequency of various trinucleotide repeats in...
journal_title：Nucleic acids research
pub_type： 杂志文章,评审
doi：10.1093/nar/gkr729
更新日期：2012-01-01 00:00:00
abstract：:Gene expression patterns have been demonstrated to be highly variable between similar cell types, for example lab strains and wild strains of Saccharomyces cerevisiae cultured under identical growth conditions exhibit a wide range of expression differences. We have used a genome-wide approach to characterize transcrip...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkj517
更新日期：2006-02-21 00:00:00
abstract：:U1 snRNP plays a crucial role in the 5' splice site recognition during splicing. Here we report the first example of naturally occurring U1-independent U2-type splicing in humans. The U1 components were not included in the pre-spliceosomal E complex formed on the human F1gamma (hF1gamma) intron 9 in vitro. Moreover, h...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gkp050
更新日期：2009-04-01 00:00:00
abstract：:The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution fol...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gku1349
更新日期：2015-01-01 00:00:00
abstract：:Rapidly evolving pathogens cause a diverse array of diseases and epidemics that threaten crop yield, food security as well as human, animal and ecosystem health. To combat infection greater comparative knowledge is required on the pathogenic process in multiple species. The Pathogen-Host Interactions database (PHI-bas...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/gku1165
更新日期：2015-01-01 00:00:00
abstract：:The -175 T greater than C mutation in the promoter of the A gamma- or G gamma-globin gene causes a 50-100 fold increase of the expression of the respective gene in adult erythroid cells (Hereditary Persistence of Fetal Hemoglobin). We show here that this mutation increases 3-9 fold the expression of a gamma-CAT report...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/17.14.5509
更新日期：1989-07-25 00:00:00
abstract：:This paper describes a computer program designed to look for similarities between pairs of nucleic or amino acid sequences. The program looks both for segments of perfect identity or for regions where, using a scoring matrix, a minimum value is exceeded. The results of comparisons are presented as a matrix which is di...
journal_title：Nucleic acids research
pub_type： 杂志文章
doi：10.1093/nar/10.9.2951
更新日期：1982-05-11 00:00:00
