作者: jumble 時(shí)間: 2025-3-21 23:07 作者: 繁重 時(shí)間: 2025-3-22 00:53
Robert D. Poodiack,William E. Woodth at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I diges作者: Muffle 時(shí)間: 2025-3-22 05:58
Robert D. Poodiack,William E. Woodf the DNA library nor the number of DNA changes has any limits. Furthermore, with the costs of synthetic DNA dropping every year, after an initial investment is made in the oligonucleotides, these can be exchanged for alternative ones with different sequences at any point in the process, fully explo作者: 拍下盜公款 時(shí)間: 2025-3-22 12:02 作者: 污點(diǎn) 時(shí)間: 2025-3-22 13:30 作者: 污點(diǎn) 時(shí)間: 2025-3-22 20:42
1064-3745 design and analysis of mutant libraries. Written in the successful .Methods in Molecular Biology. series format, chapters include introductions to their respective topics, lists o978-1-4939-5329-5978-1-4939-1053-3Series ISSN 1064-3745 Series E-ISSN 1940-6029 作者: Jogging 時(shí)間: 2025-3-22 23:48
Random Mutagenesis by Error-Prone Pol Plasmid Replication in ,nesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum 作者: 衰老 時(shí)間: 2025-3-23 05:20 作者: Irascible 時(shí)間: 2025-3-23 09:20
Restriction Enzyme-Mediated DNA Family Shufflingth at least five consecutive nucleotides of local identity. Since DNA shuffling was originally developed, many variations on the method have been published. In particular, the use of restriction enzymes in the fragmentation step allows for greater customization of fragment lengths than DNase I diges作者: 教育學(xué) 時(shí)間: 2025-3-23 12:42 作者: 令人發(fā)膩 時(shí)間: 2025-3-23 14:37 作者: Malaise 時(shí)間: 2025-3-23 21:58 作者: transdermal 時(shí)間: 2025-3-23 22:55 作者: OGLE 時(shí)間: 2025-3-24 05:42 作者: intellect 時(shí)間: 2025-3-24 08:08
Methods: Named Message Sequencescols. SeSaM is advantageous with respect to (1) elimination of mutagenic “hot spots”, (2) increase in frequency of subsequent nucleotide substitutions, (3) control over the mutational bias through the utilization of universal base analogs, and, consequently, (4) the prospect of generating transversi作者: 抑制 時(shí)間: 2025-3-24 12:37 作者: 逢迎白雪 時(shí)間: 2025-3-24 17:36 作者: Flat-Feet 時(shí)間: 2025-3-24 21:55
https://doi.org/10.1007/978-1-4302-0037-6e amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or “multisites”). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the 作者: critique 時(shí)間: 2025-3-25 01:06
Installation and Creating a Robotonal diversities while avoiding high loads of deleterious mutations. Here we describe a simple protocol for incorporating synthetic oligonucleotides that encode designed, site-specific mutations by assembly PCR. This protocol enables a researcher to “hedge the bets,” namely, to explore a large numbe作者: CLAY 時(shí)間: 2025-3-25 05:41 作者: CERE 時(shí)間: 2025-3-25 09:18 作者: Collision 時(shí)間: 2025-3-25 13:14 作者: 魯莽 時(shí)間: 2025-3-25 19:17 作者: MUTE 時(shí)間: 2025-3-25 20:06
Guglielmo Chiodi,Leonardo Dittafull randomization by making use of carefully designed mutagenic cassettes and an optimized one-pot megaprimer PCR. The method is especially suited to construct libraries of up to ten randomized codons for focused directed evolution, exhibits up to 97 % efficiency in the amplification of mutated ove作者: 冬眠 時(shí)間: 2025-3-26 00:40 作者: 防御 時(shí)間: 2025-3-26 08:23 作者: 歡樂中國 時(shí)間: 2025-3-26 09:32 作者: Embolic-Stroke 時(shí)間: 2025-3-26 13:13
https://doi.org/10.1007/978-1-4939-1053-3deletion mutations; directed evolution; gene variant libraries; insertion mutations; mutagenesis; genetic作者: 裂口 時(shí)間: 2025-3-26 18:14 作者: BUCK 時(shí)間: 2025-3-26 23:23
Elizabeth M.J. Gillam,Janine N. Copp,David AckerleIncludes cutting-edge methods and protocols.Provides step-by-step detail essential for reproducible results.Contains key notes and implementation advice from the experts作者: FLIT 時(shí)間: 2025-3-27 02:31 作者: palette 時(shí)間: 2025-3-27 08:26
Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolutiond the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present compreh作者: 情感 時(shí)間: 2025-3-27 13:20 作者: 開始從未 時(shí)間: 2025-3-27 16:44 作者: 貪婪性 時(shí)間: 2025-3-27 18:06 作者: 愛國者 時(shí)間: 2025-3-27 22:24
Generation of Effective Libraries by Neutral Drift accessible sequence space by repeated rounds of mutagenesis and selection to maintain wild-type function. Mutations that are largely neutral for the native function accumulate, and those that are highly detrimental are purged, yielding a library of high diversity and quality. This technique is usef作者: 種屬關(guān)系 時(shí)間: 2025-3-28 03:44
Site-Saturation Mutagenesis by Overlap Extension PCRorporation of degenerate base combinations at specific codon locations, giving rise to a precise series of amino acid substitutions in the encoded protein. Here we describe a simple and efficient overlap PCR protocol for the introduction of degenerate bases at either single or multiple codon locatio作者: Flawless 時(shí)間: 2025-3-28 07:48 作者: 急急忙忙 時(shí)間: 2025-3-28 11:51 作者: archaeology 時(shí)間: 2025-3-28 16:42 作者: institute 時(shí)間: 2025-3-28 19:25
Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolutioninsertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restricti作者: Galactogogue 時(shí)間: 2025-3-28 23:43
Restriction Enzyme-Mediated DNA Family Shufflingat could be accomplished using error-prone PCR alone. To achieve this, mutated copies of a protein-coding sequence are fragmented with DNase I and the fragments are then reassembled in a PCR without primers. The fragments anneal where there is sufficient sequence identity, resulting in full-length v作者: 背帶 時(shí)間: 2025-3-29 03:54
Assembly of Designed Oligonucleotides: A Useful Tool in Synthetic Biology for Creating High-Quality protein, metabolic, and genome engineering. In directed evolution of proteins, ADO benefits from using reduced amino acid alphabets for saturation mutagenesis and/or DNA shuffling, but all 20 canonical amino acids can be also used as building blocks. ADO is performed in a two-step reaction. The firs作者: 事情 時(shí)間: 2025-3-29 09:00 作者: 水土 時(shí)間: 2025-3-29 15:18
USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homolmbled into full-length genes, proven for up to ten fragments. USERec requires a minimal crossover sequence (a 5′-AN. T-3′ motif) of the fragments that can be implemented wherever structural or functional comparisons suggest a fragment boundary. The greatly reduced sequence constraints of this method作者: 緩和 時(shí)間: 2025-3-29 18:53 作者: MODE 時(shí)間: 2025-3-29 19:56 作者: insidious 時(shí)間: 2025-3-30 00:05 作者: Entreaty 時(shí)間: 2025-3-30 06:15
Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides Durinhat encode designed, site-specific mutations by assembly PCR. This protocol enables a researcher to “hedge the bets,” namely, to explore a large number of potentially beneficial mutations in a combinatorial manner such that individual library variants carry a limited number of mutations.作者: commensurate 時(shí)間: 2025-3-30 09:05 作者: inspired 時(shí)間: 2025-3-30 13:09
https://doi.org/10.1007/978-3-662-66478-0protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.作者: Condyle 時(shí)間: 2025-3-30 19:01
Methods: Named Message Sequenceson-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.作者: AVID 時(shí)間: 2025-3-31 00:37
A First Script and Its Implicationsns. The resulting libraries can then be directly screened for improved protein function as either an independent directed evolution study or an adjunct to random mutagenesis strategies (such as error-prone PCR) that are, in isolation, unlikely to access the full repertoire of possible amino acid substitutions at any given position.作者: probate 時(shí)間: 2025-3-31 01:10 作者: 遺棄 時(shí)間: 2025-3-31 06:49
https://doi.org/10.1057/9781137316837 facilitate directional assembly of gene fragments for applications such as exon or domain shuffling, loop grafting, reassembly of natural modular biosynthetic assembly lines, and rearrangement of structurally (but not sequence) homologous proteins.作者: Enrage 時(shí)間: 2025-3-31 13:12
Sraffa’s Lectures on Continental Bankingdue to changes in protein conformation and flexibility. As the effects of new termini in specific protein locations are difficult to predict, the preparation of a library constituting all possible permutation sites is an effective search strategy for identifying variants with novel properties.作者: paradigm 時(shí)間: 2025-3-31 14:47 作者: brother 時(shí)間: 2025-3-31 21:24
Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesisprotocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.作者: 易發(fā)怒 時(shí)間: 2025-3-31 23:47
The Sequence Saturation Mutagenesis (SeSaM) Methodon-enriched mutant libraries. These advanced features lead to chemically diverse mutant libraries on the protein level, essentially making SeSaM a complementary technology to transition biased epPCR mutagenesis methods.作者: ingenue 時(shí)間: 2025-4-1 01:50 作者: 肌肉 時(shí)間: 2025-4-1 08:55
Transposon-Based Approaches for Generating Novel Molecular Diversity During Directed Evolutionon endonuclease generates blunt-end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.
