Abstract:
:Model single base extension (SBE) genotyping reactions with individual deoxy-, dideoxy- and acyclonucleoside triphosphates are monitored by MALDI-TOF mass spectrometry. Three non-proofreading DNA polymerases display remarkably high misincorporation (up to 64% of correct incorporation) when extending primers with single substrates at saturating concentrations. Introduction of one phosphorothioate (PS) linkage into the primer 3' terminus reduces misincorporation by these enzymes an average 1.4-fold (range 0- to 3.5-fold) versus correct incorporation. Combined use of 3'-PS primers with strongly proofreading DNA polymerases yields order of magnitude improvements in SBE fidelity over those produced by the equivalent non-proofreading enzymes. Errors are reduced to below MALDI-TOF detectable levels in almost all cases. The Sp diastereomer of the 3'-PS primer, which can be prepared in situ by incubation with proofreading polymerase, is stable to 3'-exonuclease activity over periods longer than 16 h. Products of correct extension by T7 DNAP are retained over 30-60 min during idling turnover at a dNTP concentration of 2.5 micro M, indicating that the assay can be applied over a broad range of substrate concentrations. These results suggest that the use of PS primers and proofreading polymerases will offer a simple and cost-effective means to improve fidelity in a range of single-substrate SBE assay formats.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Di Giusto D,King GCdoi
10.1093/nar/gng007keywords:
subject
Has Abstractpub_date
2003-02-01 00:00:00pages
e7issue
3eissn
0305-1048issn
1362-4962journal_volume
31pub_type
杂志文章abstract::The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 am...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/13.21.7607
更新日期:1985-11-11 00:00:00
abstract::Replication of the promiscuous plasmid pMV158 requires expression of the initiator repB gene, which is controlled by the repressor CopG. Genes repB and copG are co-transcribed from promoter P(cr). We have studied the interactions between RNA polymerase, CopG and the promoter to elucidate the mechanism of repression by...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp503
更新日期:2009-08-01 00:00:00
abstract::Long non-coding RNAs (lncRNAs) that have no protein-coding capacity make up a large proportion of the transcriptome of various species. Many lncRNAs are expressed within the animal central nervous system in spatial- and temporal-specific patterns, indicating that lncRNAs play important roles in cellular processes, neu...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gks943
更新日期:2012-12-01 00:00:00
abstract::In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with pol...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.14.3433
更新日期:1998-07-15 00:00:00
abstract::Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formatio...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky945
更新日期:2019-01-10 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt718
更新日期:2013-11-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/28.5.1106
更新日期:2000-03-01 00:00:00
abstract::Since July 1995, the European Bioinformatics Institute (EBI) has maintained RHdb (http://www.ebi.ac.uk/RHdb/RHdb.html ), a public database for radiation hybrid data. Radiation hybrid mapping is an important technique for determining high resolution maps. Recently, CORBA access has been added to Rhdb. The EBI is an Out...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.1.102
更新日期:1998-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt335
更新日期:2013-07-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr294
更新日期:2011-07-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/10.18.5605
更新日期:1982-09-25 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt915
更新日期:2014-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv1525
更新日期:2016-05-05 00:00:00
abstract::Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gks344
更新日期:2012-08-01 00:00:00
abstract::Members of the HMG-I(Y) family of mammalian nonhistone proteins are of importance because they have been demonstrated to bind specifically to the minor groove of A.T-rich sequences both in vitro and in vivo and to function as gene transcriptional regulatory proteins in vivo. Here we report the cloning, sequencing, cha...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.18.4259
更新日期:1993-09-11 00:00:00
abstract::The rpoH genes encoding homologs of Escherichia coli sigma 32 (heat shock sigma factor) were isolated and sequenced from five gram negative proteobacteria (gamma or alpha subgroup): Enterobacter cloacae (gamma), Serratia marcescens (gamma), Proteus mirabilis (gamma), Agrobacterium tumefaciens (alpha) and Zymomonas mob...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:
更新日期:1995-11-11 00:00:00
abstract::We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinati...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn219
更新日期:2008-06-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/2.8.1381
更新日期:1975-08-01 00:00:00
abstract::Despite the large number of noncoding RNAs in human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained on...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz1192
更新日期:2020-02-20 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm824
更新日期:2007-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:1995-04-11 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/3.7.1811
更新日期:1976-07-01 00:00:00
abstract::ADGO 2.0 is a web-based tool that provides composite interpretations for microarray data comparing two sample groups as well as lists of genes from diverse sources of biological information. Some other tools also incorporate composite annotations solely for interpreting lists of genes but usually provide highly redund...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkr392
更新日期:2011-07-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky086
更新日期:2018-04-06 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw1159
更新日期:2017-01-25 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv632
更新日期:2015-11-16 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa018
更新日期:2020-03-18 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/2.12.2223
更新日期:1975-12-01 00:00:00
abstract::One of the greatest challenges that modern molecular biology is facing is the understanding of the complex mechanisms regulating gene expression. A fundamental step in this process requires the characterization of regulatory motifs playing key roles in the regulation of gene expression at transcriptional and post-tran...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkh465
更新日期:2004-07-01 00:00:00