Site-Directed Mutagenesis by Polymerase Chain Reaction View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2002

AUTHORS

Albert Jeltsch , Thomas Lanio

ABSTRACT

Site-directed mutagenesis (SDM) is a powerful tool for analyze protein structure and function, protein folding, and enzyme mechanism (1). Several protocols for SDM by polymerase chain reaction (PCR) are published (2–8). Here, two protocols, which turned out to be robust and efficient in the authors’ hands, are described in detail. Both methods comprise two steps: In the first step the desired mutation is introduced by a PCR primer used to amplify one part of the target gene. In a second step, this PCR product is then used as a megaprimer to amplify the full gene containing the mutation. Both methods deviate in how the mutated gene is introduced back into its cloning vector: In method 1, the whole vector plasmid is amplified in a PCR reaction, method 2 relies on restriction enzyme cleavage of the gene and vector, followed by ligation. In both methods, together with the mutation, a restriction enzyme marker site is introduced into the target gene by silent mutations, to allow fast and convenient screening for the presence of the mutation (4,7,9). Because random mutagenesis and directed evolution are being widely used for protein engineering (10), a method for random mutagenesis by PCR is described, using spiked oligonucleotides, which contain a mixture of all four nucleotides, to a certain degree (11). This technique allows randomization of a small part of a gene, at a level that can be chosen at will, and is a convenient alternative to cassette mutagenesis methods described previously (12,13). The protocol described here allows the construction of large mutant libraries (104-105 clones). Because wild-type (WT) alleles are efficiently excluded from transformants, screening for the presence of the mutational primer is not necessary. More... »

PAGES

85-94

Book

TITLE

In Vitro Mutagenesis Protocols

ISBN

978-0-89603-910-0
978-1-59259-194-7

Identifiers

URI

http://scigraph.springernature.com/pub.10.1385/1-59259-194-9:085

DOI

http://dx.doi.org/10.1385/1-59259-194-9:085

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1014559305


Indexing Status Check whether this publication has been indexed by Scopus and Web Of Science using the SN Indexing Status Tool
Incoming Citations Browse incoming citations for this publication using opencitations.net

JSON-LD is the canonical representation for SciGraph data.

TIP: You can open this SciGraph record using an external JSON-LD service: JSON-LD Playground Google SDTT

[
  {
    "@context": "https://springernature.github.io/scigraph/jsonld/sgcontext.json", 
    "about": [
      {
        "id": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/06", 
        "inDefinedTermSet": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/", 
        "name": "Biological Sciences", 
        "type": "DefinedTerm"
      }, 
      {
        "id": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/0601", 
        "inDefinedTermSet": "http://purl.org/au-research/vocabulary/anzsrc-for/2008/", 
        "name": "Biochemistry and Cell Biology", 
        "type": "DefinedTerm"
      }
    ], 
    "author": [
      {
        "affiliation": {
          "alternateName": "Institut f\u00fcr Biochemie, Justus-Liebig-Universit\u00e4t, Gie \u03b2en, Germany", 
          "id": "http://www.grid.ac/institutes/grid.8664.c", 
          "name": [
            "Institut f\u00fcr Biochemie, Justus-Liebig-Universit\u00e4t, Gie \u03b2en, Germany"
          ], 
          "type": "Organization"
        }, 
        "familyName": "Jeltsch", 
        "givenName": "Albert", 
        "id": "sg:person.01244204423.50", 
        "sameAs": [
          "https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01244204423.50"
        ], 
        "type": "Person"
      }, 
      {
        "affiliation": {
          "alternateName": "Institut f\u00fcr Biochemie, Justus-Liebig-Universit\u00e4t, Gie \u03b2en, Germany", 
          "id": "http://www.grid.ac/institutes/grid.8664.c", 
          "name": [
            "Institut f\u00fcr Biochemie, Justus-Liebig-Universit\u00e4t, Gie \u03b2en, Germany"
          ], 
          "type": "Organization"
        }, 
        "familyName": "Lanio", 
        "givenName": "Thomas", 
        "id": "sg:person.01346652675.62", 
        "sameAs": [
          "https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01346652675.62"
        ], 
        "type": "Person"
      }
    ], 
    "datePublished": "2002", 
    "datePublishedReg": "2002-01-01", 
    "description": "Site-directed mutagenesis (SDM) is a powerful tool for analyze protein structure and function, protein folding, and enzyme mechanism (1). Several protocols for SDM by polymerase chain reaction (PCR) are published (2\u20138). Here, two protocols, which turned out to be robust and efficient in the authors\u2019 hands, are described in detail. Both methods comprise two steps: In the first step the desired mutation is introduced by a PCR primer used to amplify one part of the target gene. In a second step, this PCR product is then used as a megaprimer to amplify the full gene containing the mutation. Both methods deviate in how the mutated gene is introduced back into its cloning vector: In method 1, the whole vector plasmid is amplified in a PCR reaction, method 2 relies on restriction enzyme cleavage of the gene and vector, followed by ligation. In both methods, together with the mutation, a restriction enzyme marker site is introduced into the target gene by silent mutations, to allow fast and convenient screening for the presence of the mutation (4,7,9). Because random mutagenesis and directed evolution are being widely used for protein engineering (10), a method for random mutagenesis by PCR is described, using spiked oligonucleotides, which contain a mixture of all four nucleotides, to a certain degree (11). This technique allows randomization of a small part of a gene, at a level that can be chosen at will, and is a convenient alternative to cassette mutagenesis methods described previously (12,13). The protocol described here allows the construction of large mutant libraries (104-105 clones). Because wild-type (WT) alleles are efficiently excluded from transformants, screening for the presence of the mutational primer is not necessary.", 
    "editor": [
      {
        "familyName": "Braman", 
        "givenName": "Jeff", 
        "type": "Person"
      }
    ], 
    "genre": "chapter", 
    "id": "sg:pub.10.1385/1-59259-194-9:085", 
    "isAccessibleForFree": false, 
    "isPartOf": {
      "isbn": [
        "978-0-89603-910-0", 
        "978-1-59259-194-7"
      ], 
      "name": "In Vitro Mutagenesis Protocols", 
      "type": "Book"
    }, 
    "keywords": [
      "site-directed mutagenesis", 
      "random mutagenesis", 
      "target genes", 
      "cassette mutagenesis method", 
      "large mutant libraries", 
      "wild-type allele", 
      "polymerase chain reaction", 
      "spiked oligonucleotides", 
      "protein folding", 
      "mutant library", 
      "protein engineering", 
      "protein structure", 
      "mutagenesis method", 
      "full gene", 
      "enzyme mechanism", 
      "mutagenesis", 
      "restriction enzyme cleavage", 
      "genes", 
      "PCR primers", 
      "vector plasmid", 
      "cloning vector", 
      "mutations", 
      "marker sites", 
      "silent mutations", 
      "enzyme cleavage", 
      "chain reaction", 
      "PCR products", 
      "PCR reaction", 
      "primers", 
      "protocol", 
      "transformants", 
      "powerful tool", 
      "nucleotides", 
      "folding", 
      "plasmid", 
      "alleles", 
      "megaprimer", 
      "cleavage", 
      "oligonucleotide", 
      "vector", 
      "convenient alternative", 
      "engineering", 
      "first step", 
      "library", 
      "sites", 
      "evolution", 
      "mechanism", 
      "presence", 
      "reaction", 
      "step", 
      "method", 
      "function", 
      "second step", 
      "authors' hands", 
      "small part", 
      "relies", 
      "technique", 
      "part", 
      "structure", 
      "levels", 
      "Method 1", 
      "ligation", 
      "certain degree", 
      "alternative", 
      "products", 
      "tool", 
      "mixture", 
      "detail", 
      "hand", 
      "construction", 
      "degree", 
      "randomization", 
      "will"
    ], 
    "name": "Site-Directed Mutagenesis by Polymerase Chain Reaction", 
    "pagination": "85-94", 
    "productId": [
      {
        "name": "dimensions_id", 
        "type": "PropertyValue", 
        "value": [
          "pub.1014559305"
        ]
      }, 
      {
        "name": "doi", 
        "type": "PropertyValue", 
        "value": [
          "10.1385/1-59259-194-9:085"
        ]
      }
    ], 
    "publisher": {
      "name": "Springer Nature", 
      "type": "Organisation"
    }, 
    "sameAs": [
      "https://doi.org/10.1385/1-59259-194-9:085", 
      "https://app.dimensions.ai/details/publication/pub.1014559305"
    ], 
    "sdDataset": "chapters", 
    "sdDatePublished": "2022-10-01T06:58", 
    "sdLicense": "https://scigraph.springernature.com/explorer/license/", 
    "sdPublisher": {
      "name": "Springer Nature - SN SciGraph project", 
      "type": "Organization"
    }, 
    "sdSource": "s3://com-springernature-scigraph/baseset/20221001/entities/gbq_results/chapter/chapter_39.jsonl", 
    "type": "Chapter", 
    "url": "https://doi.org/10.1385/1-59259-194-9:085"
  }
]
 

Download the RDF metadata as:  json-ld nt turtle xml License info

HOW TO GET THIS DATA PROGRAMMATICALLY:

JSON-LD is a popular format for linked data which is fully compatible with JSON.

curl -H 'Accept: application/ld+json' 'https://scigraph.springernature.com/pub.10.1385/1-59259-194-9:085'

N-Triples is a line-based linked data format ideal for batch operations.

curl -H 'Accept: application/n-triples' 'https://scigraph.springernature.com/pub.10.1385/1-59259-194-9:085'

Turtle is a human-readable linked data format.

curl -H 'Accept: text/turtle' 'https://scigraph.springernature.com/pub.10.1385/1-59259-194-9:085'

RDF/XML is a standard XML format for linked data.

curl -H 'Accept: application/rdf+xml' 'https://scigraph.springernature.com/pub.10.1385/1-59259-194-9:085'


 

This table displays all metadata directly associated to this object as RDF triples.

139 TRIPLES      22 PREDICATES      98 URIs      91 LITERALS      7 BLANK NODES

Subject Predicate Object
1 sg:pub.10.1385/1-59259-194-9:085 schema:about anzsrc-for:06
2 anzsrc-for:0601
3 schema:author N22e1555b95e240dd9fbe11984b95442f
4 schema:datePublished 2002
5 schema:datePublishedReg 2002-01-01
6 schema:description Site-directed mutagenesis (SDM) is a powerful tool for analyze protein structure and function, protein folding, and enzyme mechanism (1). Several protocols for SDM by polymerase chain reaction (PCR) are published (2–8). Here, two protocols, which turned out to be robust and efficient in the authors’ hands, are described in detail. Both methods comprise two steps: In the first step the desired mutation is introduced by a PCR primer used to amplify one part of the target gene. In a second step, this PCR product is then used as a megaprimer to amplify the full gene containing the mutation. Both methods deviate in how the mutated gene is introduced back into its cloning vector: In method 1, the whole vector plasmid is amplified in a PCR reaction, method 2 relies on restriction enzyme cleavage of the gene and vector, followed by ligation. In both methods, together with the mutation, a restriction enzyme marker site is introduced into the target gene by silent mutations, to allow fast and convenient screening for the presence of the mutation (4,7,9). Because random mutagenesis and directed evolution are being widely used for protein engineering (10), a method for random mutagenesis by PCR is described, using spiked oligonucleotides, which contain a mixture of all four nucleotides, to a certain degree (11). This technique allows randomization of a small part of a gene, at a level that can be chosen at will, and is a convenient alternative to cassette mutagenesis methods described previously (12,13). The protocol described here allows the construction of large mutant libraries (104-105 clones). Because wild-type (WT) alleles are efficiently excluded from transformants, screening for the presence of the mutational primer is not necessary.
7 schema:editor N6f942d8ba5614be8912b25b6377171e5
8 schema:genre chapter
9 schema:isAccessibleForFree false
10 schema:isPartOf N63f6fbd6bc8a4e9e8289df5764b81394
11 schema:keywords Method 1
12 PCR primers
13 PCR products
14 PCR reaction
15 alleles
16 alternative
17 authors' hands
18 cassette mutagenesis method
19 certain degree
20 chain reaction
21 cleavage
22 cloning vector
23 construction
24 convenient alternative
25 degree
26 detail
27 engineering
28 enzyme cleavage
29 enzyme mechanism
30 evolution
31 first step
32 folding
33 full gene
34 function
35 genes
36 hand
37 large mutant libraries
38 levels
39 library
40 ligation
41 marker sites
42 mechanism
43 megaprimer
44 method
45 mixture
46 mutagenesis
47 mutagenesis method
48 mutant library
49 mutations
50 nucleotides
51 oligonucleotide
52 part
53 plasmid
54 polymerase chain reaction
55 powerful tool
56 presence
57 primers
58 products
59 protein engineering
60 protein folding
61 protein structure
62 protocol
63 random mutagenesis
64 randomization
65 reaction
66 relies
67 restriction enzyme cleavage
68 second step
69 silent mutations
70 site-directed mutagenesis
71 sites
72 small part
73 spiked oligonucleotides
74 step
75 structure
76 target genes
77 technique
78 tool
79 transformants
80 vector
81 vector plasmid
82 wild-type allele
83 will
84 schema:name Site-Directed Mutagenesis by Polymerase Chain Reaction
85 schema:pagination 85-94
86 schema:productId N9456250eb9004fd59859f2dd675a74a6
87 Nfc6f4c3771514f1ea58151e7ecc6ed5a
88 schema:publisher Nca6c6ded552949c7b6e32331d66e6566
89 schema:sameAs https://app.dimensions.ai/details/publication/pub.1014559305
90 https://doi.org/10.1385/1-59259-194-9:085
91 schema:sdDatePublished 2022-10-01T06:58
92 schema:sdLicense https://scigraph.springernature.com/explorer/license/
93 schema:sdPublisher N9f455cb488c44869be739422b7507333
94 schema:url https://doi.org/10.1385/1-59259-194-9:085
95 sgo:license sg:explorer/license/
96 sgo:sdDataset chapters
97 rdf:type schema:Chapter
98 N22e1555b95e240dd9fbe11984b95442f rdf:first sg:person.01244204423.50
99 rdf:rest Na8bb82750dad479cbc046bc70af12b3c
100 N63f6fbd6bc8a4e9e8289df5764b81394 schema:isbn 978-0-89603-910-0
101 978-1-59259-194-7
102 schema:name In Vitro Mutagenesis Protocols
103 rdf:type schema:Book
104 N6f942d8ba5614be8912b25b6377171e5 rdf:first N95135f2724344bd9881d7d2a663631ab
105 rdf:rest rdf:nil
106 N9456250eb9004fd59859f2dd675a74a6 schema:name doi
107 schema:value 10.1385/1-59259-194-9:085
108 rdf:type schema:PropertyValue
109 N95135f2724344bd9881d7d2a663631ab schema:familyName Braman
110 schema:givenName Jeff
111 rdf:type schema:Person
112 N9f455cb488c44869be739422b7507333 schema:name Springer Nature - SN SciGraph project
113 rdf:type schema:Organization
114 Na8bb82750dad479cbc046bc70af12b3c rdf:first sg:person.01346652675.62
115 rdf:rest rdf:nil
116 Nca6c6ded552949c7b6e32331d66e6566 schema:name Springer Nature
117 rdf:type schema:Organisation
118 Nfc6f4c3771514f1ea58151e7ecc6ed5a schema:name dimensions_id
119 schema:value pub.1014559305
120 rdf:type schema:PropertyValue
121 anzsrc-for:06 schema:inDefinedTermSet anzsrc-for:
122 schema:name Biological Sciences
123 rdf:type schema:DefinedTerm
124 anzsrc-for:0601 schema:inDefinedTermSet anzsrc-for:
125 schema:name Biochemistry and Cell Biology
126 rdf:type schema:DefinedTerm
127 sg:person.01244204423.50 schema:affiliation grid-institutes:grid.8664.c
128 schema:familyName Jeltsch
129 schema:givenName Albert
130 schema:sameAs https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01244204423.50
131 rdf:type schema:Person
132 sg:person.01346652675.62 schema:affiliation grid-institutes:grid.8664.c
133 schema:familyName Lanio
134 schema:givenName Thomas
135 schema:sameAs https://app.dimensions.ai/discover/publication?and_facet_researcher=ur.01346652675.62
136 rdf:type schema:Person
137 grid-institutes:grid.8664.c schema:alternateName Institut für Biochemie, Justus-Liebig-Universität, Gie βen, Germany
138 schema:name Institut für Biochemie, Justus-Liebig-Universität, Gie βen, Germany
139 rdf:type schema:Organization
 




Preview window. Press ESC to close (or click here)


...