612:
31:
433:
504:
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.
428:
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
583:
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
500:
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
574:
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
464:
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
205:
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
649:
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.
451:
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
563:. 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.
496:. 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
376:
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
297:
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.
306:
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
139:
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
520:) 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.
501:
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.
420:
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.
254:
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
78:
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
559:
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
1018:
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".
634:
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
214:. 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.
615:
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.
436:
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.
645:(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
285:
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
991:
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".
70:
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
809:
575:
of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.
158:
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
399:
Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to
290:
596:
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
190:. 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
460:
series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy numbers, for example,
722:
852:
767:
294:
1177:
1150:
413:
of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.
174:
All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a
541:
201:
instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this
641:, whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate
588:(SV40) was used in first cloning experiment involving mammalian cells. A number of vectors based on other type of viruses like
529:
378:
116:
1195:"Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells"
791:
123:, for example very large DNA fragments, and other organisms such as yeast may be used. Cloning vectors in yeast include
1306:
79:
664:
571:
556:
537:
207:
124:
1311:
659:
67:
335:
227:
183:
597:
243:
365:
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Some vectors are designed for transcription only with no heterologous protein expressed, for example for
250:. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and
327:
267:
175:
151:
146:. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in
50:
that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for
1061:
913:"A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene"
1206:
601:
560:
489:
89:
There are many types of cloning vectors, but the most commonly used ones are genetically engineered
679:
1043:
893:
638:
477:
361:
211:
179:
75:
516:
are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (
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1234:
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1035:
1000:
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cells. Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a
393:
369:
351:
315:
223:
136:
51:
1140:
839:
751:
742:
186:-amplified target gene also digested with the same enzymes is ligated into the vectors using
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1111:
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1027:
963:
924:
877:
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401:
95:
71:
1254:"A new generation of human artificial chromosomes for functional genomics and gene therapy"
868:
Romanos MA, Scorer CA, Clare JJ (June 1992). "Foreign gene expression in yeast: a review".
406:
357:
1193:
Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, et al. (December 2011).
472:
origin of replication and may be used to generate single-stranded DNA. These are called
1210:
381:
so that proteins are only produced when required. Some commonly used promoters are the
1278:
1253:
1229:
1194:
446:
331:
235:
163:
59:
1116:
1091:
1300:
928:
605:
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198:
182:. The restriction sites in the MCS are first cleaved by restriction enzymes, then a
104:
1047:
897:
589:
410:
259:
202:
108:
698:
1107:
82:. After a DNA fragment has been cloned into a cloning vector, it may be further
759:
319:
263:
191:
1199:
Proceedings of the
National Academy of Sciences of the United States of America
17:
1269:
1031:
611:
386:
339:
308:
247:
239:
231:
187:
392:. The presence of a promoter is necessary when screening techniques such as
1219:
493:
251:
83:
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63:
1142:
Molecular
Biotechnology Principles and Applications of Recombinant DNA
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951:
334:. Examples of fusion partners that may be used for screening are the
282:
which are used with their corresponding auxotrophic strains of yeast.
674:
453:
429:
cloning large fragments may require more specialised cloning vector.
255:
35:
912:
38:
plasmid, one of the first plasmids widely used as a cloning vector.
1069:
642:
631:
610:
461:
457:
431:
155:
55:
29:
585:
432:
278:
266:
selection markers that allow an auxotrophic organism to grow in
1252:
Kouprina N, Earnshaw WC, Masumoto H, Larionov V (April 2013).
373:
226:
is carried by the vector to allow the selection of positively
47:
911:
Yazynin SA, Deyev SM, Jucovic M, Hartley RW (February 1996).
356:
A cloning vector need not contain suitable elements for the
1090:
Chauthaiwale VM, Therwath A, Deshpande VV (December 1992).
847:. Methods in Molecular Biology. Vol. 235. p. 23.
119:(BACs). Some DNA, however, cannot be stably maintained in
536:
with a copy number of only 1 per cell. BACs are based on
234:
resistance is often used as marker, an example being the
750:. Methods in Molecular Biology. Vol. 498. pp.
74:. The vector and the foreign DNA may be treated with a
54:
purposes. The cloning vector may be DNA taken from a
86:
into another vector designed for more specific use.
748:
High
Throughput Protein Expression and Purification
741:
792:"Cloning Methods - Recombination cloning systems"
368:(RBS), many however do, and may then work as an
178:(MCS) or polylinker, which contains many unique
952:"Positive selection of recombinant DNA by CcdB"
206:without the use of DNA digest and ligase is by
1169:Gene Cloning and DNA Analysis: An Introduction
93:. Cloning is generally first performed using
833:
831:
829:
827:
825:
823:
528:Insert size of up to 350 kb can be cloned in
8:
488:The bacteriophages used for cloning are the
456:plasmid. Other cloning vectors include the
810:"Gateway® Recombination Cloning Technology"
740:Esposito D, Garvey LA, Chakiath CS (2009).
620:Screening: example of the blue/white screen
540:, another artificial chromosome called the
1092:"Bacteriophage lambda as a cloning vector"
1277:
1228:
1218:
1115:
967:
743:"Gateway cloning for protein expression"
270:may also be used; examples of these are
262:are effective in different cell types.
690:
238:gene, which confers resistance to the
62:of a higher organism, or it may be the
723:"The Technology Behind TOPO® Cloning"
630:Many general purpose vectors such as
608:have been used with limited success.
135:All commonly used cloning vectors in
7:
1258:Cellular and Molecular Life Sciences
360:of a cloned target gene, such as a
194:into another vector if necessary.
25:
1172:. Wiley-Blackwell. p. 100.
330:to facilitate the production of
117:bacterial artificial chromosomes
34:Schematic representation of the
1139:Glick BR, Pasternak JJ (2005).
530:bacterial artificial chromosome
524:Bacterial artificial chromosome
326:in frame with and flanking the
162:, and these vectors are called
27:Small piece of maintainable DNA
699:"Definition of cloning vector"
579:Animal and plant viral vectors
532:(BAC). BACs are maintained in
197:Other cloning vectors may use
1:
210:, for example as used in the
1108:10.1128/mr.56.4.577-591.1992
929:10.1016/0378-1119(95)00814-4
838:Casali N, Preston A (2003).
131:Features of a cloning vector
125:yeast artificial chromosomes
1145:(3rd ed.). ASM Press.
760:10.1007/978-1-59745-196-3_3
665:Plant transformation vector
572:Human artificial chromosome
567:Human artificial chromosome
557:Yeast artificial chromosome
552:Yeast artificial chromosome
480:series of cloning vectors.
1338:
660:Vector (molecular biology)
623:
444:
349:
99:, and cloning vectors in
1270:10.1007/s00018-012-1113-3
1032:10.1007/s10529-004-3518-z
950:Bernard P (August 1996).
468:Some plasmids contain an
336:green fluorescent protein
314:for α complementation in
598:Cauliflower mosaic virus
424:Types of cloning vectors
1220:10.1073/pnas.1114483108
1166:TA Brown (2010-04-19).
1096:Microbiological Reviews
1066:Genetics Institute, Inc
476:, and examples are the
346:Elements for expression
244:beta-lactam antibiotics
150:, such as a functional
616:
437:
366:ribosomal binding site
212:Gateway cloning system
39:
1020:Biotechnology Letters
882:10.1002/yea.320080602
614:
435:
268:minimal growth medium
176:multiple cloning site
152:origin of replication
33:
602:Tobacco mosaic virus
561:Human Genome Project
394:blue-white selection
316:blue-white selection
66:of a bacterium. The
46:is a small piece of
1307:Genetics techniques
1211:2011PNAS..10820048K
1205:(50): 20048–20053.
680:Golden Gate Cloning
617:
438:
103:include plasmids,
80:molecular ligation
76:restriction enzyme
40:
1312:Molecular biology
1026:(21): 1659–1663.
969:10.2144/96212pf01
854:978-1-58829-151-6
769:978-1-58829-879-9
703:Genome Dictionary
670:IMAGE cDNA clones
626:Blue white screen
470:M13 bacteriophage
370:expression vector
352:Expression vector
224:selectable marker
218:Selectable marker
208:DNA recombination
180:restriction sites
137:molecular biology
72:restriction sites
16:(Redirected from
1329:
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1264:(7): 1135–1148.
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1068:. Archived from
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584:vector based on
544:is based on the
96:Escherichia coli
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639:β-galactosidase
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594:Papilloma virus
586:Simian virus 40
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486:
449:
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332:fusion proteins
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220:
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28:
23:
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18:Cloning vectors
15:
12:
11:
5:
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1179:978-1444334074
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1102:(4): 577–591.
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999:(4): 784–788.
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876:(6): 423–488.
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606:Gemini viruses
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465:to the cells.
447:Plasmid vector
445:Main article:
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372:. The target
350:Main article:
347:
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324:reporter genes
303:
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236:beta-lactamase
219:
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171:
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164:shuttle vector
132:
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105:bacteriophages
44:cloning vector
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1152:9781555816124
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1072:on 2013-04-19
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1062:"Copy number"
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1074:. Retrieved
1070:the original
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706:. Retrieved
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590:Adenoviruses
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411:cDNA library
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309:
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260:hygromycin B
221:
203:TOPO cloning
196:
173:
170:Cloning site
159:
154:(ori). The
147:
141:
134:
120:
100:
94:
88:
43:
41:
478:pBluescript
320:marker gene
264:Auxotrophic
228:transformed
192:sub-cloning
1301:Categories
1076:2013-03-06
814:Invitrogen
727:Invitrogen
708:2012-10-18
686:References
396:are used.
358:expression
340:luciferase
338:(GFP) and
312:α fragment
293:, and the
248:ampicillin
240:penicillin
232:Antibiotic
188:DNA ligase
538:F plasmid
494:M13 phage
474:phagemids
390:promoters
379:inducible
318:, and/or
295:parD/parE
252:kanamycin
242:group of
107:(such as
84:subcloned
1322:Plasmids
1288:22907415
1239:22123967
1048:10312859
1040:15604816
1005:10769758
898:15674832
778:18988017
654:See also
546:P1 phage
498:lysogeny
418:in vitro
362:promoter
230:cells.
127:(YACs).
91:plasmids
1317:Cloning
1279:3522797
1230:3250132
1207:Bibcode
1126:1480110
978:8862819
937:8635737
890:1502852
842:E. coli
636:E. coli
534:E. coli
514:Cosmids
490:λ phage
441:Plasmid
407:genomic
402:E. coli
287:barnase
160:E. coli
148:E. coli
143:E. coli
121:E. coli
113:cosmids
109:phage λ
101:E. coli
64:plasmid
52:cloning
1286:
1276:
1237:
1227:
1176:
1149:
1124:
1117:372889
1114:
1046:
1038:
1003:
976:
935:
896:
888:
851:
776:
766:
675:fosmid
509:Cosmid
454:pBR322
256:zeocin
115:, and
68:vector
58:, the
36:pBR322
1044:S2CID
894:S2CID
870:Yeast
752:31–54
647:lacZα
643:X-gal
632:pUC19
462:pUC19
246:like
156:ColE1
56:virus
1284:PMID
1235:PMID
1174:ISBN
1147:ISBN
1122:PMID
1036:PMID
1001:PMID
974:PMID
933:PMID
917:Gene
886:PMID
849:ISBN
796:EMBL
774:PMID
764:ISBN
604:and
592:and
492:and
385:and
364:and
310:lacZ
291:Ccda
279:URA3
276:and
273:LEU2
258:and
60:cell
1274:PMC
1266:doi
1225:PMC
1215:doi
1203:108
1112:PMC
1104:doi
1028:doi
964:doi
925:doi
921:169
878:doi
756:doi
542:PAC
518:cos
458:pUC
409:or
388:lac
374:DNA
328:MCS
322:or
184:PCR
111:),
48:DNA
1303::
1282:.
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1262:70
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1022:.
997:28
995:.
972:.
960:21
958:.
954:.
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884:.
872:.
822:^
812:.
794:.
772:.
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548:.
383:T7
342:.
289:,
222:A
166:.
42:A
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20:)
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