601:
20:
422:
493:
There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.
417:
A large number of cloning vectors are available, and choosing the vector may depend upon a number of factors, such as the size of the insert, copy number and cloning method. Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore
572:
Viruses that infect plant and animal cells have also been manipulated to introduce foreign genes into plant and animal cells. The natural ability of viruses to adsorb to cells, introduce their DNA and replicate have made them ideal vehicles to transfer foreign DNA into eukaryotic cells in culture. A
489:
since using phage λ as a cloning vector involves only the lytic cycle. There are two kinds of λ phage vectors - insertion vector and replacement vector. Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. In replacement vectors, the cleavage
563:
may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem
453:
has a copy number of 500-700 copies per cell, and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic
194:
method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning
638:
sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.
440:
Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA inserts of up to 15 kb in size. One of the earliest commonly used cloning vectors is the
552:. It contains a telomeric sequence, an autonomously replicating sequence (features required to replicate linear chromosomes in yeast cells). These vectors also contain suitable restriction sites to clone foreign DNA as well as genes to be used as selectable markers.
485:. There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for
365:
may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host. Where the promoter is present, the expression of the gene is preferably tightly controlled and
286:
toxins. This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.
295:
Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such features present in cloning vectors may be the
128:
have key features necessary for their function, such as a suitable cloning site and selectable marker. Others may have additional features specific to their use. For reason of ease and convenience, cloning is often performed using
509:) which contains elements required for packaging DNA into λ particles. Under apt origin of replication (ori), it can replicate as a plasmid It is normally used to clone large DNA fragments between 28 and 45 Kb.
490:
sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.
409:
mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.
243:
resistance gene. Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to
67:
that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by
548:
are used as vectors to clone DNA fragments of more than 1 mega base (1Mb=1000kb) in size. They are useful in cloning larger DNA fragments as required in mapping genomes such as in the
1007:
Kim HG, Kim HS, Hwang HJ, Chung SK, Lee JM, Chung DK (November 2004). "Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products".
623:
usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for
203:. The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.
604:
An LB agar plate showing the result of a blue white screen. White colonies may contain an insert in the plasmid it carries, while the blue ones are unsuccessful clones.
425:
The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for blue-white screen.
634:(5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based
274:
Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as
980:
Gabant P, Van Reeth T, Drèze PL, Faelen M, Szpirer C, Szpirer J (April 2000). "New positive selection system based on the parD (kis/kid) system of the R1 plasmid".
59:
contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of
798:
564:
of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.
147:
origin of replication is found in many plasmids. Some vectors also include elements that allow them to be maintained in another organism in addition to
388:
Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to
585:
have been used to clone genes in mammals. At present, retroviral vectors are popular for cloning genes in mammalian cells. In case of plants like
179:. The target DNA sequence can be inserted into the vector in a specific direction if so desired. The restriction sites may be further used for
449:
series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy numbers, for example,
711:
466:
402:
of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.
279:
163:
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a
190:
instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this
630:, whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate
577:(SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like
1184:"Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells"
841:
780:
756:
283:
112:, for example very large DNA fragments, and other organisms such as yeast may be used. Cloning vectors in yeast include
1166:
1139:
530:
518:
367:
105:
1295:
68:
653:
560:
545:
526:
405:
Some vectors are designed for transcription only with no heterologous protein expressed, for example for
239:. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and
196:
113:
135:. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in
39:
that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for
1300:
1050:
902:"A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene"
648:
56:
324:
216:
172:
78:
There are many types of cloning vectors, but the most commonly used ones are genetically engineered
586:
232:
505:
are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (
354:
1156:
394:
cells. Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a
1129:
828:
740:
731:
316:
256:
175:-amplified target gene also digested with the same enzymes is ligated into the vectors using
164:
140:
1243:"A new generation of human artificial chromosomes for functional genomics and gene therapy"
1195:
857:
Romanos MA, Scorer CA, Clare JJ (June 1992). "Foreign gene expression in yeast: a review".
590:
549:
478:
8:
1182:
Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, et al. (December 2011).
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origin of replication and may be used to generate single-stranded DNA. These are called
1199:
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so that proteins are only produced when required. Some commonly used promoters are the
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171:. The restriction sites in the MCS are first cleaved by restriction enzymes, then a
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71:. After a DNA fragment has been cloned into a cloning vector, it may be further
748:
1188:
Proceedings of the
National Academy of Sciences of the United States of America
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320:
224:
152:
48:
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381:. The presence of a promoter is necessary when screening techniques such as
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Molecular
Biotechnology Principles and Applications of Recombinant DNA
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940:
323:. Examples of fusion partners that may be used for screening are the
271:
which are used with their corresponding auxotrophic strains of yeast.
482:
418:
cloning large fragments may require more specialised cloning vector.
240:
72:
901:
534:
486:
462:
371:
27:
plasmid, one of the first plasmids widely used as a cloning vector.
1058:
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261:
131:
101:
79:
52:
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selection markers that allow an auxotrophic organism to grow in
663:
442:
244:
24:
19:
1241:
Kouprina N, Earnshaw WC, Masumoto H, Larionov V (April 2013).
608:
215:
is carried by the vector to allow the selection of positively
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900:
Yazynin SA, Deyev SM, Jucovic M, Hartley RW (February 1996).
631:
620:
450:
446:
144:
44:
345:
A cloning vector need not contain suitable elements for the
1079:
Chauthaiwale VM, Therwath A, Deshpande VV (December 1992).
836:. Methods in Molecular Biology. Vol. 235. p. 23.
574:
267:
108:(BACs). Some DNA, however, cannot be stably maintained in
979:
899:
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with a copy number of only 1 per cell. BACs are based on
362:
223:
resistance is often used as marker, an example being the
36:
1181:
739:. Methods in Molecular Biology. Vol. 498. pp.
421:
63:. The vector and the foreign DNA may be treated with a
728:
43:
purposes. The cloning vector may be DNA taken from a
75:
into another vector designed for more specific use.
737:
High
Throughput Protein Expression and Purification
856:
730:
512:
781:"Cloning Methods - Recombination cloning systems"
567:
357:(RBS), many however do, and may then work as an
1287:
1006:
167:(MCS) or polylinker, which contains many unique
941:"Positive selection of recombinant DNA by CcdB"
195:without the use of DNA digest and ligase is by
119:
1158:Gene Cloning and DNA Analysis: An Introduction
1127:
555:
540:
82:. Cloning is generally first performed using
826:
822:
820:
818:
816:
814:
812:
517:Insert size of up to 350 kb can be cloned in
477:The bacteriophages used for cloning are the
445:plasmid. Other cloning vectors include the
799:"Gateway® Recombination Cloning Technology"
729:Esposito D, Garvey LA, Chakiath CS (2009).
609:Screening: example of the blue/white screen
529:, another artificial chromosome called the
412:
1081:"Bacteriophage lambda as a cloning vector"
809:
334:
1266:
1217:
1207:
1104:
956:
1154:
732:"Gateway cloning for protein expression"
599:
420:
259:may also be used; examples of these are
251:are effective in different cell types.
18:
938:
227:gene, which confers resistance to the
51:of a higher organism, or it may be the
1288:
712:"The Technology Behind TOPO® Cloning"
619:Many general purpose vectors such as
597:have been used with limited success.
124:All commonly used cloning vectors in
1247:Cellular and Molecular Life Sciences
206:
349:of a cloned target gene, such as a
13:
183:into another vector if necessary.
14:
1322:
1161:. Wiley-Blackwell. p. 100.
472:
319:to facilitate the production of
290:
106:bacterial artificial chromosomes
23:Schematic representation of the
1234:
1175:
1148:
1128:Glick BR, Pasternak JJ (2005).
1121:
1072:
1043:
1000:
973:
519:bacterial artificial chromosome
513:Bacterial artificial chromosome
315:in frame with and flanking the
158:
151:, and these vectors are called
16:Small piece of maintainable DNA
932:
893:
850:
791:
773:
722:
704:
688:"Definition of cloning vector"
680:
568:Animal and plant viral vectors
521:(BAC). BACs are maintained in
186:Other cloning vectors may use
1:
674:
199:, for example as used in the
1097:10.1128/mr.56.4.577-591.1992
918:10.1016/0378-1119(95)00814-4
827:Casali N, Preston A (2003).
120:Features of a cloning vector
114:yeast artificial chromosomes
7:
1134:(3rd ed.). ASM Press.
749:10.1007/978-1-59745-196-3_3
654:Plant transformation vector
642:
561:Human artificial chromosome
556:Human artificial chromosome
546:Yeast artificial chromosome
541:Yeast artificial chromosome
469:series of cloning vectors.
10:
1327:
649:Vector (molecular biology)
612:
433:
429:
338:
88:, and cloning vectors in
1259:10.1007/s00018-012-1113-3
1021:10.1007/s10529-004-3518-z
939:Bernard P (August 1996).
497:
457:Some plasmids contain an
325:green fluorescent protein
303:for α complementation in
587:Cauliflower mosaic virus
413:Types of cloning vectors
1209:10.1073/pnas.1114483108
1155:TA Brown (2010-04-19).
1085:Microbiological Reviews
1055:Genetics Institute, Inc
465:, and examples are the
335:Elements for expression
233:beta-lactam antibiotics
139:, such as a functional
605:
426:
355:ribosomal binding site
201:Gateway cloning system
28:
1009:Biotechnology Letters
871:10.1002/yea.320080602
603:
424:
257:minimal growth medium
165:multiple cloning site
141:origin of replication
22:
591:Tobacco mosaic virus
550:Human Genome Project
383:blue-white selection
305:blue-white selection
55:of a bacterium. The
35:is a small piece of
1296:Genetics techniques
1200:2011PNAS..10820048K
1194:(50): 20048–20053.
669:Golden Gate Cloning
606:
427:
92:include plasmids,
69:molecular ligation
65:restriction enzyme
29:
1301:Molecular biology
1015:(21): 1659–1663.
958:10.2144/96212pf01
843:978-1-58829-151-6
758:978-1-58829-879-9
692:Genome Dictionary
659:IMAGE cDNA clones
615:Blue white screen
459:M13 bacteriophage
359:expression vector
341:Expression vector
213:selectable marker
207:Selectable marker
197:DNA recombination
169:restriction sites
126:molecular biology
61:restriction sites
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1057:. Archived from
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573:vector based on
533:is based on the
85:Escherichia coli
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583:Papilloma virus
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1168:978-1444334074
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1091:(4): 577–591.
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988:(4): 784–788.
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436:Plasmid vector
434:Main article:
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361:. The target
339:Main article:
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313:reporter genes
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225:beta-lactamase
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153:shuttle vector
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94:bacteriophages
33:cloning vector
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1061:on 2013-04-19
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1063:. Retrieved
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695:. Retrieved
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579:Adenoviruses
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400:cDNA library
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249:hygromycin B
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192:TOPO cloning
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159:Cloning site
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143:(ori). The
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130:
123:
109:
89:
83:
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32:
30:
467:pBluescript
309:marker gene
253:Auxotrophic
217:transformed
181:sub-cloning
1290:Categories
1065:2013-03-06
803:Invitrogen
716:Invitrogen
697:2012-10-18
675:References
385:are used.
347:expression
329:luciferase
327:(GFP) and
301:α fragment
282:, and the
237:ampicillin
229:penicillin
221:Antibiotic
177:DNA ligase
527:F plasmid
483:M13 phage
463:phagemids
379:promoters
368:inducible
307:, and/or
284:parD/parE
241:kanamycin
231:group of
96:(such as
73:subcloned
1311:Plasmids
1277:22907415
1228:22123967
1037:10312859
1029:15604816
994:10769758
887:15674832
767:18988017
643:See also
535:P1 phage
487:lysogeny
407:in vitro
351:promoter
219:cells.
116:(YACs).
80:plasmids
1306:Cloning
1268:3522797
1219:3250132
1196:Bibcode
1115:1480110
967:8862819
926:8635737
879:1502852
831:E. coli
625:E. coli
523:E. coli
503:Cosmids
479:λ phage
430:Plasmid
396:genomic
391:E. coli
276:barnase
149:E. coli
137:E. coli
132:E. coli
110:E. coli
102:cosmids
98:phage λ
90:E. coli
53:plasmid
41:cloning
1275:
1265:
1226:
1216:
1165:
1138:
1113:
1106:372889
1103:
1035:
1027:
992:
965:
924:
885:
877:
840:
765:
755:
664:fosmid
498:Cosmid
443:pBR322
245:zeocin
104:, and
57:vector
47:, the
25:pBR322
1033:S2CID
883:S2CID
859:Yeast
741:31–54
636:lacZα
632:X-gal
621:pUC19
451:pUC19
235:like
145:ColE1
45:virus
1273:PMID
1224:PMID
1163:ISBN
1136:ISBN
1111:PMID
1025:PMID
990:PMID
963:PMID
922:PMID
906:Gene
875:PMID
838:ISBN
785:EMBL
763:PMID
753:ISBN
593:and
581:and
481:and
374:and
353:and
299:lacZ
280:Ccda
268:URA3
265:and
262:LEU2
247:and
49:cell
1263:PMC
1255:doi
1214:PMC
1204:doi
1192:108
1101:PMC
1093:doi
1017:doi
953:doi
914:doi
910:169
867:doi
745:doi
531:PAC
507:cos
447:pUC
398:or
377:lac
363:DNA
317:MCS
311:or
173:PCR
100:),
37:DNA
1292::
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1251:70
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537:.
372:T7
331:.
278:,
211:A
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31:A
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