108:
Consequently, the fully randomised 'NNN' is not ideal, and alternative, more restricted degenerate codons are used. 'NNK' and 'NNS' have the benefit of encoding all 20 amino acids, but still encode a stop codon 3% of the time. Alternative codons such as 'NDT', 'DBK' avoid stop codons entirely, and encode a minimal set of amino acids that still encompass all the main biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small). In the case there is no restriction to use a single degenerate codon only, it is possible to reduce the bias considerably. Several computational tools were developed to allow high level of control over the degenerate codons and their corresponding amino acids.
80:
28:
83:
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).
107:
Different degenerate codons can be used to encode sets of amino acids. Because some amino acids are encoded by more codons than others, the exact ratio of amino acids cannot be equal. Additionally, it is usual to use degenerate codons that minimise stop codons (which are generally not desired).
31:
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 at position 5 are shown
406:
Kille, Sabrina; Acevedo-Rocha, Carlos G.; Parra, Loreto P.; Zhang, Zhi-Gang; Opperman, Diederik J.; Reetz, Manfred T.; Acevedo, Juan Pablo (2013-02-15). "Reducing Codon
Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis".
362:
Reetz, Manfred T.; Prasad, Shreenath; Carballeira, José D.; Gumulya, Yosephine; Bocola, Marco (2010-07-07). "Iterative
Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods".
84:
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).
645:
Shivange, Amol V; Marienhagen, Jan; Mundhada, Hemanshu; Schenk, Alexander; Schwaneberg, Ulrich (2009-02-01). "Advances in generating functional diversity for directed protein evolution".
610:
Chica, Robert A.; et al. (2005). "Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design".
534:
Engqvist, Martin K. M.; Nielsen, Jens (2015-04-30). "ANT: Software for
Generating and Evaluating Degenerate Codons for Natural and Expanded Genetic Codes".
44:
491:
Halweg-Edwards, Andrea L.; Pines, Gur; Winkler, James D.; Pines, Assaf; Gill, Ryan T. (September 16, 2016). "A Web
Interface for Codon Compression".
444:"MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites"
262:
Reetz, M. T.; Carballeira J. D. (2007). "Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes".
237:
67:(performing a site saturation at every site in the protein, resulting in a library of size that contains every possible
686:
79:
691:
97:
89:
569:
Kell, Douglas B.; Day, Philip J.; Breitling, Rainer; Green, Lucy; Currin, Andrew; Swainston, Neil (2017-07-10).
114:
696:
48:
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225:
662:
627:
592:
551:
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473:
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388:
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27:
654:
619:
582:
543:
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372:
334:
318:
271:
17:
442:
Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui (June 2014).
68:
339:
306:
680:
291:
623:
59:
at the position. There are many variants of the site saturation technique, from
658:
547:
504:
307:"An efficient one-step site-directed and site-saturation mutagenesis protocol"
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469:
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330:
666:
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348:
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587:
570:
322:
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443:
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93:
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52:
26:
224:
Saturation mutagenesis is commonly used to generate variants for
305:
Zheng, Lei; Baumann, Ulrich; Reymond, Jean-Louis (2004-07-15).
649:. Biocatalysis and Biotransformation/Bioinorganic Chemistry.
63:(saturating two positions in every mutant in the library) to
571:"CodonGenie: optimised ambiguous codon design tools"
55:or set of codons is substituted with all possible
92:PCR with a randomised codon in the primers (e.g.
88:Saturation mutagenesis is commonly achieved by
8:
586:
459:
338:
365:Journal of the American Chemical Society
110:
249:
104:used at the codons to be randomised.
7:
454:(6): 301–302, 304, 306–308, passim.
257:
255:
253:
647:Current Opinion in Chemical Biology
25:
37:Site saturation mutagenesis (SSM)
612:Current Opinion in Biotechnology
238:Sequence Saturation Mutagenesis
100:, with a mixture of synthesis
1:
624:10.1016/j.copbio.2005.06.004
18:Site saturation mutagenesis
713:
659:10.1016/j.cbpa.2009.01.019
548:10.1021/acssynbio.5b00018
505:10.1021/acssynbio.6b00026
98:artificial gene synthesis
90:site-directed mutagenesis
65:scanning site saturation
575:PeerJ Computer Science
311:Nucleic Acids Research
85:
61:paired site saturation
33:
536:ACS Synthetic Biology
493:ACS Synthetic Biology
409:ACS Synthetic Biology
276:10.1038/nprot.2007.72
82:
30:
588:10.7717/peerj-cs.120
128:Amino acids encoded
51:, in which a single
687:Genetics techniques
122:No. of amino acids
49:protein engineering
692:Molecular genetics
323:10.1093/nar/gnh110
226:directed evolution
86:
47:technique used in
45:random mutagenesis
34:
461:10.2144/000114177
421:10.1021/sb300037w
377:10.1021/ja1030479
371:(26): 9144–9152.
217:
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71:of the protein).
16:(Redirected from
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671:
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642:
636:
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607:
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566:
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531:
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499:(9): 1021–1023.
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264:Nature Protocols
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115:Degenerate codon
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21:
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41:site saturation
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15:
12:
11:
5:
710:
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673:
672:
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618:(4): 378–384.
602:
561:
542:(8): 935–938.
526:
483:
434:
398:
354:
297:
270:(4): 891–903.
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119:No. of codons
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24:
14:
13:
10:
9:
6:
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3:
2:
709:
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629:
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448:BioTechniques
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196:ARCGILMFSTWV
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179:RNDCGHILFSYV
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125:No. of stops
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81:
74:
72:
70:
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62:
58:
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50:
46:
42:
38:
29:
19:
653:(1): 19–25.
650:
646:
640:
615:
611:
605:
578:
574:
564:
539:
535:
529:
496:
492:
486:
451:
447:
437:
415:(2): 83–92.
412:
408:
401:
368:
364:
357:
317:(14): e115.
314:
310:
300:
267:
263:
223:
220:Applications
106:
87:
69:point mutant
64:
60:
40:
39:, or simply
36:
35:
697:Mutagenesis
102:nucleotides
57:amino acids
681:Categories
244:References
150:NNK / NNS
597:2376-5992
513:2161-5063
470:1940-9818
385:0002-7863
331:0305-1048
213:RNDCGHSY
667:19261539
632:15994074
581:: e120.
556:25901796
521:27169595
478:24924390
429:23656371
393:20536132
349:15304544
292:37361631
284:17446890
232:See also
96:) or by
162:All 20
145:All 20
43:, is a
665:
630:
595:
554:
519:
511:
476:
468:
427:
391:
383:
347:
340:514394
337:
329:
290:
282:
75:Method
32:below.
288:S2CID
94:SeSaM
53:codon
663:PMID
628:PMID
593:ISSN
552:PMID
517:PMID
509:ISSN
474:PMID
466:ISSN
425:PMID
389:PMID
381:ISSN
345:PMID
327:ISSN
280:PMID
201:NRT
184:DBK
167:NDT
133:NNN
655:doi
620:doi
583:doi
544:doi
501:doi
456:doi
417:doi
373:doi
369:132
335:PMC
319:doi
272:doi
190:12
187:18
173:12
170:12
156:20
153:32
139:20
136:64
683::
661:.
651:13
626:.
616:16
614:.
591:.
577:.
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550:.
538:.
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507:.
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464:.
452:56
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315:32
313:.
309:.
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252:^
228:.
210:0
207:8
204:8
193:0
176:0
159:1
142:3
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657::
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622::
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585::
579:3
558:.
546::
540:4
523:.
503::
497:5
480:.
458::
431:.
419::
413:2
395:.
375::
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321::
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274::
268:2
20:)
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