Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round

Seyfang, Andreas; Jian, Jin

doi:10.1016/j.ab.2003.10.012

Cited by 47 publications

(61 citation statements)

References 21 publications

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“…We wished to allow multiple mutations per clone, both because this allowed for greater mutational coverage for any given library size, and offered an opportunity to discover intragenic epistatic relationships. To this end, we scaled up a previous mutagenesis protocol (Seyfang & Huaqian Jin, 2004) to develop Precision Oligo‐Pool based Code Alteration (POPCode), which yields random codon replacements (see Materials and Methods). …”

Section: Resultsmentioning

confidence: 99%

“…The Precision Oligo‐Pool based Code Alteration (POPCode) scales up a previous method (Seyfang & Huaqian Jin, 2004) to achieve coverage over the complete spectrum of possible amino acid changes at all protein positions. POPCode requires design of an oligonucleotide centered on each codon in the open reading frame (ORF) of interest, such that the target codon is replaced with an NNK degenerate codon.…”

Section: Methodsmentioning

confidence: 99%

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A framework for exhaustively mapping functional missense variants

Weile

Sun

Coté

et al. 2017

Molecular Systems Biology

165

286

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Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin‐like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A framework for exhaustively mapping functional missense variants

Weile

Sun

Coté

et al. 2017

Molecular Systems Biology

165

286

View full text Add to dashboard Cite

show abstract

“…Multiple point mutations (TAG 3 CAG) in intronic sequences were introduced as described in ref. 53, and single point mutations were also created as described (29). Precise intron deletion mutations (⌬int6 and ⌬int7) were created by back-to-back PCR (29).…”

Section: Methodsmentioning

confidence: 99%

An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

Proc. Natl. Acad. Sci. U.S.A.

115

109

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The neurodegenerative disease spinal muscular atrophy is caused by mutation of the survival motor neuron 1 (SMN1) gene. SMN2 is a nearly identical copy of SMN1 that is unable to prevent disease, because most SMN2 transcripts lack exon 7 and thus produce a nonfunctional protein. A key cause of inefficient SMN2 exon 7 splicing is a single nucleotide difference between SMN1 and SMN2 within exon 7. We previously provided evidence that this base change suppresses exon 7 splicing by creating an inhibitory element, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer. We now find that another rare nucleotide difference between SMN1 and SMN2, in intron 7, potentially creates a second SMN2-specific hnRNP A1 binding site. Remarkably, this single base change does indeed create a highaffinity hnRNP A1 binding site, and base substitutions that disrupt it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding in vitro. We propose that interactions between hnRNP A1 molecules bound to the exonic and intronic sites cooperate to exclude exon 7 and discuss the significance of this exclusion with respect to SMN expression and splicing control more generally.alternative splicing ͉ exonic splicing silencer ͉ hnRNP A1 ͉ intronic splicing silencer A lternative splicing of mRNA precursors is an important mechanism that regulates gene expression and generates increased protein diversity from a limited number of genes. Indeed, expression of 70% or more of human genes is now estimated to involve alternative splicing (1, 2). In higher eukaryotes, alternative splicing plays an essential role in diverse cellular functions, contributing to such basic processes as cell growth, differentiation, and cell death (3, 4). Several types of cis-acting RNA elements and trans-acting protein factors help to regulate alternative splicing and do so by diverse mechanisms. In general, however, non-spliceosomal proteins that participate in regulating alternative splicing associate with regulatory elements in exons and/or introns. Serine/arginine-rich proteins (5) are typically involved in positive regulation of splicing, stimulating splicing by interacting with exonic splicing enhancer elements (ESEs) or intronic splicing enhancer elements. In contrast, negative regulation is promoted most frequently by heterogeneous nuclear ribonucleoproteins (hnRNPs) (6), which function by binding sequences known as exonic splicing silencers (ESSs) or intronic splicing silencers.Several hnRNP proteins have been identified as key splicing repressors. Among these, the abundant hnRNP A1 protein has been extensively characterized. The first hnRNP A1-dependent ESS was identified in studies of HIV tat exon 2 repression (7-9). This ESS was found to bind hnRNP A1, and mutations disrupting the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing. Several mechanisms have been proposed to explain hnRNP A1-mediated splicing repression. In one, hnRNP A1 binds to an ESS and through direct protein-protein interactions, recruits more ...

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“…As stated previously, SDM plays an important role in genetic research, such as understanding the regulation site of operon or the relationship of protein structure to function (Ling and Robinson, 1997;Seyfang and Jin, 2004;Wan et al, 2012). Therefore, many strategies used for Single-SDM are springing up with the development of genetic engineering technology (see Section 2.1).…”

Section: Strategies Of Multi-sdmmentioning

confidence: 99%

“…It plays a great role in understanding the regulatory motifs of operon and the relationship of protein structure to function (Ling and Robinson, 1997;Seyfang and Jin, 2004;Wu et al, 2013). Depending on the numbers of mutational sites, SDM can be divided into two types: single site-directed mutagenesis (Single-SDM) and multiple site-directed mutagenesis (Multi-SDM) (Liang et al, 2012).…”

Section: Strategies For Sdmmentioning

confidence: 99%

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Zhang

2016

J. Zhejiang Univ. Sci. B

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Abstract:With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

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Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round

Cited by 47 publications

References 21 publications

A framework for exhaustively mapping functional missense variants

A framework for exhaustively mapping functional missense variants

An intronic element contributes to splicing repression in spinal muscular atrophy

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

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