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excision involves subcloning often using traditional restriction enzymes and cloning strategies. In vitro excision can be more time-consuming and may require more "hands-on" work than in vivo excision systems. In either case, the systems allow the movement of the vector from the phage into a live cell, where the vector can replicate and propagate until the library is to be used.
249:. It thus represents the genes that were being actively transcribed in that particular source under the physiological, developmental, or environmental conditions that existed when the mRNA was purified. cDNA libraries can be generated using techniques that promote "full-length" clones or under conditions that generate shorter fragments used for the identification of "
143:. This image shows the saturation mutagenesis of a single position in a theoretical 10-residue protein. The wild type version of the protein is shown at the top, with M representing the first amino acid methionine, and * representing the termination of translation. All 19 mutants of the isoleucine at position 5 are shown below.
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Vectors are propagated most commonly in bacterial cells, but if using a YAC (Yeast
Artificial Chromosome) then yeast cells may be used. Vectors could also be propagated in viruses, but this can be time-consuming and tedious. However, the high transfection efficiency achieved by using viruses (often
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cDNA libraries require care to ensure that full length clones of mRNA are captured as cDNA (which will later be inserted into vectors). Several protocols have been designed to optimise the synthesis of the 1st cDNA strand and the 2nd cDNA strand for this reason, and also to make directional cloning
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sample sequence space. The amino acid substituted into a given position is shown. Each dot or set of connected dots is one member of the library. Error-prone PCR randomly mutates some residues to other amino acids. Alanine scanning replaces each residue of the protein with alanine, one-by-one. Site
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Depiction of one common way to clone a site-directed mutagenesis library (i.e., using degenerate oligos). The gene of interest is PCRed with oligos that contain a region that is perfectly complementary to the template (blue), and one that differs from the template by one or more nucleotides (red).
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Additionally, for cDNA libraries, a system using the Lambda Zap II phage, ExAssist, and 2 E. coli species has been developed. A Cre-Lox system using loxP sites and the in vivo expression of the recombinase enzyme can also be used instead. These are examples of in vivo excision systems. In vitro
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proteins from these libraries can then be screened for variants which exhibit favorable properties (e.g. stability, binding affinity or enzyme activity). This can be repeated in cycles of creating gene variants and screening the expression products in a
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is a set of clones that together represents the entire genome of a given organism. The number of clones that constitute a genomic library depends on (1) the size of the genome in question and (2) the insert size tolerated by the particular
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Many such primers containing degeneracy in the non-complementary region are pooled into the same PCR, resulting in many different PCR products with different mutations in that region (individual mutants shown with different colors below).
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or BAC library) or yeast such that each organism contains on average one construct (vector + insert). As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated (thus, "cloned").
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If creating an mRNA library (i.e. with cDNA clones), there are several possible protocols for isolating full length mRNA. To extract DNA for genomic DNA (also known as gDNA) libraries, a DNA mini-prep may be useful.
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and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of
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The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules.
188:(formed from genomic DNA) and randomized mutant libraries (formed by de novo gene synthesis where alternative nucleotides or codons are incorporated). DNA library technology is a mainstay of current
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In contrast to the library types described above, a variety of artificial methods exist for making libraries of variant genes. Variation throughout the gene can be introduced randomly by either
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purified from a particular source (either a collection of cells, a particular tissue, or an entire organism), which has been converted back to a DNA template by the use of the enzyme
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system. For most practical purposes, the tissue source of the genomic DNA is unimportant because each cell of the body contains virtually identical DNA (with some exceptions).
772:
Wang, Tian-Wen; Zhu, Hu; Ma, Xing-Yuan; Zhang, Ting; Ma, Yu-Shu; Wei, Dong-Zhi (2006-09-01). "Mutant library construction in directed molecular evolution".
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Crameri A, Raillard SA, Bermudez E, Stemmer WP (January 1998). "DNA shuffling of a family of genes from diverse species accelerates directed evolution".
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cDNA libraries are useful in reverse genetics, but they only represent a very small (less than 1%) portion of the overall genome in a given organism.
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McCullum, Elizabeth O.; Williams, Berea A. R.; Zhang, Jinglei; Chaput, John C. (2010), Braman, Jeff (ed.), "Random
Mutagenesis by Error-Prone PCR",
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phages) makes them useful for packaging the vector (with the ligated insert) and then introducing them into the bacterial (or yeast) cell.
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of a gene in a controlled way. This results in a mixture of double stranded DNA molecules which represent variants of the original gene.
725:"Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion"
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200:, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the
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Wajapeyee, Narendra; Liu, Alex Y.; Forloni, Matteo (2018-03-01). "Random
Mutagenesis Using Error-Prone DNA Polymerases".
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saturation substitutes each of the 20 possible amino acids (or some subset of them) at a single position, one-by-one.
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gDNA fragments are generated from the extracted gDNA by using non-specific frequent cutter restriction enzymes.
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Study of the repertoire of mRNAs expressed in different cells or tissues
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The nucleotide sequences of interest are preserved as inserts to a
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Determining the complete genome sequence of a given organism (see
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60:. Unsourced material may be challenged and removed.
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406:Overview of cDNA library preparation techniques
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546:that has been used to infect bacterial cells.
634:In Vitro Mutagenesis Protocols: Third Edition
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307:Applications of genomic libraries include:
266:Cloning of full-length cDNA molecules for
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259:Applications of cDNA libraries include:
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241:represents a sample of the
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263:Discovery of novel genes
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251:expressed sequence tags
182:reverse-transcribed RNA
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384:saturation mutagenesis
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278:alternative splicing
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194:genetic engineering
168:is a collection of
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