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genetic change for the whole organism and subsequent generations. This "germ line gene therapy" is considered by many to be unethical in human beings. The second type of gene therapy, "somatic cell gene therapy", is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.
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trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.
587:, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme so that colonies containing transformed DNA remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.
1932:
90:). This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large number of bacteria, each of which contains copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking,
31:
594:. Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained. This may be accomplished through a very wide range of experimental methods, including the use of
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643:, and how that expression is related to other processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to
123:, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme,
558:. Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid sequences will die. When mammalian cells (e.g. human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the
131:. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972.
663:
Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein. In practice, it is frequently more difficult to develop an organism that produces an active form of the recombinant protein in desirable
542:
Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which
225:
Notably, the growing capacity and fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These projects may include very long strands of novel DNA sequence and/or test entire libraries simultaneously, as opposed to of individual sequences. These shifts
176:
Before actual cloning experiments are performed in the lab, most cloning experiments are planned in a computer, using specialized software. Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.g. PCR primer design, dedicated software exist
631:
Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned
221:
In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into
105:
Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. There is also a lower chance of success
735:
Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy
731:
Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent
491:
The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen
63:
refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will
118:
Prior to the 1970s, the understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular
622:
Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science. A few of the more important applications are
533:
involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient
482:
DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting in a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself,
297:
and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a
313:
is chosen that contains appropriate signals for transcription and translation in the desired host organism. Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed
378:
The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see
387:
and this restriction enzyme was isolated from E.coli. Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site) and are located within a gene (frequently
447:). cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined by the designer. Such a designed sequence may be required when moving genes across
158:
Molecular cloning is similar to PCR in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an
582:
to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of
71:
In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with
392:) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (
483:
vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.
1712:
428:), as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography).
470:
The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are simply mixed together at appropriate concentrations and exposed to an enzyme
523:. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.
217:
The overall goal of molecular cloning is to take a gene of interest from one plasmid and insert it into another plasmid. This is done by performing PCR, digestive reaction, ligation reaction, and transformation.
691:, used to treat strokes), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g.
664:
quantities than it is to clone the gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.
396:) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.
458:
The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (
1742:
543:
cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.
1008:"Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli"
279:
Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium
82:
bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are
309:
Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an
143:
is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for
325:
Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:
703:
Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed
102:
and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.
226:
introduce complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction. Examples of such tools are
1435:
Wirth R, Friesenegger A, Fiedler S (Mar 1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation".
151:
is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the
76:
to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of
1828:
424:
For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from
318:) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by
222:
host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts and biological properties.
433:
383:). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme or restriction endonuclease, for example
1602:"Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. Taiwan Childhood Hepatoma Study Group"
711:), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (
68:
for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
1237:
Higuchi R, Bowman B, Freiberger M, Ryder OA, Wilson AC (1984). "DNA sequences from the quagga, an extinct member of the horse family".
671:. These include--(1) medically useful proteins whose administration can correct a defective or poorly expressed gene (e.g. recombinant
17:
1912:
1821:
607:
1091:"Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector"
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enzyme that builds the DNA chain. The mix goes through cycles of heating and cooling to produce large quantities of copied DNA.
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refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a
1968:
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uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast,
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when inserting large-sized DNA sequences. Inserts larger than 10kbp have very limited success, but bacteriophages such as
1814:
1800:
479:. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.
462:) containing desired restriction sites may be added to create end structures that are compatible with the vector.
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299:
704:
511:
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed
87:
912:
Nathans D, Smith HO (1975). "Restriction endonucleases in the analysis and restructuring of dna molecules".
1958:
1784:
688:
668:
476:
202:
1963:
1936:
303:
1388:"The transformation of genetics by DNA: an anniversary celebration of Avery, MacLeod and McCarty (1944)"
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333:
is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism
590:
The total population of individual clones obtained in a molecular cloning experiment is often termed a
73:
1879:
707:(GMOs). Although most GMOs are generated for purposes of basic biological research (see for example,
648:
640:
603:
512:
493:
444:
429:
409:
194:
519:. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed
1288:
Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang X, et al. (November 2016).
1902:
530:
497:
1065:
416:(the building blocks of DNA), primers (short pieces of complementary single stranded DNA) and a
268:
253:
1642:
1210:
515:, and cells that are in a physiological state such that they can take up DNA are said to be
451:(for example, from the mitochondria to the nucleus) or simply for increasing expression via
1806:
1246:
1102:
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960:
516:
393:
569:
8:
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687:), (2) proteins that can be administered to assist in a life-threatening emergency (e.g.
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gene that can be used to enable the survival of cells that have taken up vector sequences
127:, fragments generated by restriction enzymes could be joined in new combinations, termed
1290:"Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant"
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1387:
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DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (
198:
120:
1779:
1042:
1007:
983:
948:
695:), and (4) recombinant proteins as standard material for diagnostic laboratory tests.
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provide a means to screen for genes on the basis of the expressed protein's function.
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mechanisms through which bacteria restricted the growth of bacteriophage, isolated
1684:
1089:
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (Sep 1992).
234:
1597:
1403:
636:
526:
505:
475:) that covalently links the ends together. This joining reaction is often termed
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148:
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128:
50:
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1618:
1601:
1894:
1593:
1589:
1095:
Proceedings of the
National Academy of Sciences of the United States of America
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Proceedings of the
National Academy of Sciences of the United States of America
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Proceedings of the
National Academy of Sciences of the United States of America
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65:
54:
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1952:
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1165:
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213:
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Molecular cloning takes advantage of the fact that the chemical structure of
1567:
1115:
1032:
973:
883:(Fourth ed.). Belmont, CA, Brooks/Cole: Cengage Learning. p. 380.
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1183:
599:
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Lewandowski C, Barsan W (Feb 2001). "Treatment of acute ischemic stroke".
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1266:
1134:
1051:
992:
369:. The DNA is cleaved at the palindromic sequence to produce 'sticky ends'.
1305:
933:
672:
591:
1339:"Synonymous but not the same: the causes and consequences of codon bias"
635:
At the level of individual genes, molecular clones are used to generate
1907:
1846:
1448:
783:
Molecular
Biotechnology: Principles and Applications of Recombinant DNA
676:
647:
the genes, or make more subtle mutations using regional mutagenesis or
570:
Screening for clones with desired DNA inserts and biological properties
555:
551:
472:
432:(PCR) methods are often used for amplification of specific DNA or RNA (
413:
319:
260:
152:
124:
83:
1729:
1713:"Why Johnny can't clone: Common pitfalls and not so common solutions"
1258:
949:"Construction of biologically functional bacterial plasmids in vitro"
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537:
486:
1354:
354:
gene that can be used to screen for cells containing the foreign DNA
1671:
Pfeifer A, Verma IM (2001). "Gene therapy: promises and problems".
684:
632:
fragments of the genome, and assembling the overlapping sequences.
161:
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for the purpose. Software for the purpose include for example ApE
1527:
The MJ (Nov 1989). "Human insulin: DNA technology's first drug".
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720:
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99:
78:
59:
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can be modified to successfully insert a sequence up to 40 kbp.
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425:
249:
30:
465:
1150:"Structure and function of type II restriction endonucleases"
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384:
363:
239:
1836:
1236:
231:
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1216:. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory.
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and later derivatives including the pGEM vectors) use the
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244:
140:
1434:
187:
34:
Diagram of molecular cloning using bacteria and plasmids
1088:
946:
1596:, Lai MS, Hsu HM, Wu TC, Kong MS, Liang DC, Shau WY,
947:
Cohen SN, Chang AC, Boyer HW, Helling RB (Nov 1973).
675:, a blood-clotting factor deficient in some forms of
658:
546:
When bacterial cells are used as host organisms, the
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to serve as sites where foreign DNA may be introduced
1203:
1201:
1199:
1197:
1195:
1193:
780:
252:. It's a circular piece of DNA 4361 bases long. Two
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816:
814:
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810:
808:
806:
804:
802:
751:
749:
443:
or cDNA cloning), or in the form of synthetic DNA (
1477:
1209:
758:Recombinant DNA: genes and genomes: a short course
538:Selection of organisms containing vector sequences
487:Introduction of recombinant DNA into host organism
399:
1553:
1190:
1005:
850:(Fifth ed.). Brooks/Cole, Cengage Learning.
1950:
1066:"plasmid / plasmids | Learn Science at Scitable"
799:
774:
746:
667:Many useful proteins are currently available as
550:is usually a gene that confers resistance to an
408:DNA for cloning is most commonly produced using
205:, etc., that is, all the steps described below.
1666:
1664:
1588:
1207:
562:cassette) confers resistance to the antibiotic
554:that would otherwise kill the cells, typically
193:(commercial). These programs allow to simulate
1480:"Haemophilia care then, now and in the future"
1147:
878:
845:
823:Gene cloning and DNA analysis: an introduction
1822:
1647:. Vol. 40. Academic Press. p. 508.
1673:Annual Review of Genomics and Human Genetics
1670:
1661:
1547:
1471:
1336:
1330:
911:
373:
248:Diagram of a commonly used cloning plasmid;
1582:
1520:
1428:
1379:
1230:
1082:
651:. Genes cloned into expression vectors for
466:Creation of recombinant DNA with DNA ligase
1829:
1815:
1006:Jackson DA, Symons RH, Berg P (Oct 1972).
639:that are used for examining how genes are
362:Cleavage of a DNA sequence containing the
338:restriction endonuclease recognition sites
240:Choice of host organism and cloning vector
233:(free for academia) or GeneticConstructor
1728:
1710:
1617:
1495:
1478:Oldenburg J, Dolan G, Lemm G (Jan 2009).
1411:
1385:
1362:
1313:
1173:
1124:
1114:
1041:
1031:
982:
972:
905:
781:Patten CL, Glick BR, Pasternak J (2009).
1913:Restriction fragment length polymorphism
436:) sequences prior to molecular cloning.
403:
357:
243:
212:
86:or genetically modified microorganisms (
29:
1148:Pingoud A, Jeltsch A (September 2001).
698:
627:Genome organization and gene expression
574:Modern bacterial cloning vectors (e.g.
14:
1951:
1640:
1212:Molecular cloning: a laboratory manual
755:
259:are present, conferring resistance to
1810:
1529:American Journal of Hospital Pharmacy
820:
1337:Plotkin JB, Kudla G (January 2011).
41:is a set of experimental methods in
1606:The New England Journal of Medicine
1526:
926:10.1146/annurev.bi.44.070175.001421
27:Set of methods in molecular biology
24:
1703:
659:Production of recombinant proteins
25:
1980:
1759:
719:, and fluorescent tropical fish (
1931:
1930:
1794:
1497:10.1111/j.1365-2516.2008.01946.x
1437:Molecular & General Genetics
825:. Cambridge, MA: Blackwell Pub.
534:methods to move DNA into cells.
165:solution, free of living cells.
1634:
1281:
1208:Russell DW, Sambrook J (2001).
1141:
879:Garrett RH, Grisham CM (2010).
846:Garrett RH, Grisham CM (2013).
785:. Washington, D.C.: ASM Press.
760:. San Francisco: W.H. Freeman.
726:
717:herbicide-resistant crop plants
617:
400:Preparation of DNA to be cloned
300:bacterial artificial chromosome
94:refers to DNA molecules, while
1711:Matsumura I (September 2015).
1058:
999:
940:
872:
839:
705:genetically modified organisms
683:, used to treat some forms of
49:molecules and to direct their
13:
1:
1685:10.1146/annurev.genom.2.1.177
914:Annual Review of Biochemistry
739:
608:restriction fragment analysis
412:. Template DNA is mixed with
183:(open source), Serial Cloner
1969:Molecular biology techniques
1785:Resources in other libraries
1556:Annals of Emergency Medicine
689:tissue plasminogen activator
7:
1619:10.1056/NEJM199706263362602
596:nucleic acid hybridizations
580:blue-white screening system
322:into a specialized vector.
304:yeast artificial chromosome
134:
10:
1985:
1404:10.1093/genetics/136.2.423
723:) for home entertainment.
180:(open source), DNAStrider
113:
45:that are used to assemble
18:Recombinant DNA technology
1926:
1893:
1880:Site-directed mutagenesis
1845:
1780:Resources in your library
649:site-directed mutagenesis
604:polymerase chain reaction
445:artificial gene synthesis
430:Polymerase chain reaction
374:Preparation of vector DNA
345:selectable genetic marker
121:restriction endonucleases
1386:Lederberg J (Feb 1994).
1343:Nature Reviews. Genetics
306:vector is often chosen.
208:
1568:10.1067/mem.2001.111573
1116:10.1073/pnas.89.18.8794
1033:10.1073/pnas.69.10.2904
974:10.1073/pnas.70.11.3240
172:cloning and simulations
1840:: key methods of study
1294:Nucleic Acids Research
1166:10.1093/nar/29.18.3705
1154:Nucleic Acids Research
421:
370:
291:plasmid cloning vector
276:
271:that the host uses to
236:(free for academics).
218:
57:. The use of the word
35:
1535:(11 Suppl 2): S9-11.
407:
361:
269:origin of replication
254:antibiotic resistance
247:
216:
33:
1803:at Wikimedia Commons
699:Transgenic organisms
669:recombinant products
394:alkaline phosphatase
186:(gratis), Collagene
147:, and the resulting
64:serve as the living
1959:Genetics techniques
1855:Gel electrophoresis
1251:1984Natur.312..282H
1107:1992PNAS...89.8794S
1024:1972PNAS...69.2904J
965:1973PNAS...70.3240C
693:hepatitis B vaccine
336:one or more unique
199:restriction digests
189:(open source), and
1964:Molecular genetics
1875:Restriction digest
1838:Molecular genetics
1641:August JT (1997).
1449:10.1007/BF00332248
1306:10.1093/nar/gkw756
756:Watson JD (2007).
679:, and recombinant
653:functional cloning
585:beta-galactosidase
453:codon optimization
422:
390:beta-galactosidase
371:
331:replication origin
277:
219:
36:
1946:
1945:
1860:Molecular cloning
1801:Molecular cloning
1799:Media related to
1771:Molecular cloning
1766:Library resources
1730:10.2144/000114324
1654:978-0-08-058132-3
1300:(19): 9342–9357.
1245:(5991): 282–284.
1223:978-0-87969-576-7
1160:(18): 3705–3727.
890:978-0-495-10935-8
857:978-1-133-10629-6
832:978-1-4051-1121-8
792:978-1-55581-498-4
767:978-0-7167-2866-5
623:summarized here.
548:selectable marker
441:complementary DNA
311:expression vector
96:molecular cloning
43:molecular biology
39:Molecular cloning
16:(Redirected from
1976:
1934:
1933:
1870:Promoter bashing
1831:
1824:
1817:
1808:
1807:
1798:
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1750:
1741:. Archived from
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1490:(Suppl 1): 2–7.
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869:
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837:
836:
821:Brown T (2006).
818:
797:
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778:
772:
771:
753:
709:transgenic mouse
367:restriction site
286:Escherichia coli
21:
1984:
1983:
1979:
1978:
1977:
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1973:
1949:
1948:
1947:
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1922:
1903:Gene sequencing
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1704:Further reading
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747:
742:
729:
701:
661:
629:
620:
600:antibody probes
572:
540:
527:Electroporation
506:electroporation
489:
468:
426:extinct animals
402:
376:
242:
211:
174:
149:recombinant DNA
145:DNA replication
137:
129:recombinant DNA
116:
108:bacteriophage λ
92:recombinant DNA
47:recombinant DNA
28:
23:
22:
15:
12:
11:
5:
1982:
1972:
1971:
1966:
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1895:Bioinformatics
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1760:External links
1758:
1756:
1755:
1723:(3): IV–XIII.
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1101:(18): 8794–7.
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1070:www.nature.com
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612:DNA sequencing
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513:transformation
494:transformation
488:
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418:DNA polymerase
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316:shuttle vector
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55:host organisms
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1745:on 2015-09-16
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449:genetic codes
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19:
1935:
1918:STR analysis
1859:
1847:Experimental
1770:
1747:. Retrieved
1743:the original
1720:
1716:
1676:
1672:
1644:Gene Therapy
1643:
1636:
1609:
1605:
1600:(Jun 1997).
1584:
1559:
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1532:
1528:
1522:
1487:
1483:
1473:
1443:(1): 175–7.
1440:
1436:
1430:
1398:(2): 423–6.
1395:
1391:
1381:
1349:(1): 32–42.
1346:
1342:
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1073:. Retrieved
1069:
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881:Biochemistry
880:
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848:Biochemistry
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822:
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727:Gene therapy
702:
666:
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634:
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621:
618:Applications
589:
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545:
541:
531:transduction
525:
521:transfection
510:
502:transfection
498:transduction
490:
481:
469:
459:
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351:
344:
337:
330:
324:
308:
294:
284:
280:
278:
265:tetracycline
230:, Teselagen
224:
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175:
169:
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138:
117:
104:
95:
91:
77:
70:
58:
38:
37:
1679:: 177–211.
1484:Haemophilia
673:factor VIII
592:DNA library
51:replication
1953:Categories
1908:Microarray
1749:2 February
1075:2017-12-06
920:: 273–93.
740:References
677:hemophilia
645:inactivate
556:ampicillin
552:antibiotic
473:DNA ligase
320:subcloning
261:ampicillin
155:organism.
153:transgenic
125:DNA ligase
84:transgenic
74:vector DNA
899:297392560
866:777722371
641:expressed
564:Geneticin
517:competent
273:replicate
267:, and an
203:ligations
170:In silico
1937:Category
1739:26345511
1693:11701648
1590:Chang MH
1576:11174240
1514:29118026
1506:19125934
1465:25214157
1392:Genetics
1373:21102527
1324:27596602
1184:11557805
713:pharming
685:diabetes
477:ligation
289:) and a
275:the DNA.
191:SnapGene
162:in vitro
135:Overview
1628:9197213
1598:Chen DS
1594:Chen CJ
1541:2690608
1457:2659971
1422:8150273
1413:1205797
1364:3074964
1315:5100594
1275:4313241
1267:6504142
1247:Bibcode
1135:1528894
1103:Bibcode
1052:4342968
1020:Bibcode
993:4594039
961:Bibcode
721:GloFish
681:insulin
610:and/or
460:linkers
381:DNA end
295:E. coli
281:E. coli
228:GenoCAD
114:History
100:plasmid
79:E. coli
60:cloning
53:within
1768:about
1737:
1691:
1651:
1626:
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1512:
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1455:
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1371:
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1239:Nature
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1043:389671
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991:
984:427208
981:
934:166604
932:
897:
887:
864:
854:
829:
789:
764:
637:probes
492:(e.g.
434:RT-PCR
250:pBR322
1510:S2CID
1461:S2CID
1271:S2CID
1175:55916
1126:50007
576:pUC19
560:kanMX
414:bases
385:EcoRI
364:BamHI
257:genes
209:Steps
1751:2016
1735:PMID
1689:PMID
1649:ISBN
1624:PMID
1572:PMID
1537:PMID
1502:PMID
1453:PMID
1418:PMID
1369:PMID
1320:PMID
1263:PMID
1218:ISBN
1180:PMID
1131:PMID
1048:PMID
989:PMID
930:PMID
895:OCLC
885:ISBN
862:OCLC
852:ISBN
827:ISBN
787:ISBN
762:ISBN
329:DNA
263:and
88:GMOs
66:host
1725:doi
1681:doi
1614:doi
1610:336
1564:doi
1492:doi
1445:doi
1441:216
1408:PMC
1400:doi
1396:136
1359:PMC
1351:doi
1310:PMC
1302:doi
1255:doi
1243:312
1170:PMC
1162:doi
1121:PMC
1111:doi
1038:PMC
1028:doi
979:PMC
969:doi
922:doi
715:),
508:).
410:PCR
352:tag
302:or
141:DNA
1955::
1733:.
1721:59
1719:.
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1675:.
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471:(
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