Cloning

from Wikipedia, the free encyclopedia

Cloning (or cloning , English molecular cloning ) is the umbrella term in molecular biology for methods of obtaining and identical duplication of deoxyribonucleic acid (DNA). In contrast to cloning , the aim of which is to produce genetically identical organisms, cloning is limited to producing identical molecules of DNA.

properties

During cloning, a desired DNA fragment (for example a gene or the cDNA coding for a protein ) is integrated into a vector (for example a plasmid or viral vector ). The aim of cloning is to multiply a DNA fragment in order to investigate its properties or to use it in further processes. After a replication, a multiple of the amount of DNA used initially can be obtained by isolating the plasmid DNA, which, in contrast to in vitro methods such as PCR, is inexpensive, precise and in large numbers. Alternatively, the cells can be a gene product , such as recombinant proteins expressing (for example, at a protein overexpression). Cloning plays a role

Various organisms are used as hosts for the replication of the cloning products. Well-known examples are bacterial cells such as the Escherichia coli bacterium , unicellular algae or fungi. The host cells multiply by cell division , with identical copies of the target DNA to be cloned being produced. The result is a population of cells that all contain a clone of the desired DNA fragment. A suitable clone is isolated from this population for further use.

The cloned DNA can be used to transfer one or more genes to foreign organisms in order to improve metabolic processes or to confer resistance (genetic manipulation in animals and plants). By a functional cloning similar DNA sequences are identified by a positional cloning adjacent DNA sequences are identified.

Procedure

So-called vectors (“gene ferries”) are used during cloning . These serve as a means of transport for the transfer of a specific DNA sequence (called transgene or insert ) into a recipient cell and its replication. There are various methods to make these vectors receptive to foreign DNA:

Restriction and ligation

Schematic representation of a cloning with restriction and ligation.

In the plasmid cloning a plasmid (e.g., pUC19 ) in the course of a restriction digestion using specific restriction enzymes cut offset so that overhanging ends (engl. Sticky ends , sticky end) occur. The DNA to be inserted is a fragment that was isolated from another vector using the same enzymes, synthetic DNA with the appropriate overhangs or DNA amplified from genomic DNA or cDNA using the polymerase chain reaction (PCR) . Before the PCR products are inserted into the vector, they are cut with the same enzymes so that complementary ends are created on the vector and target DNA. The mutually compatible, overhanging ends of vector and target DNA are found and hybridize with one another. In the subsequent ligation , which is catalyzed by a DNA ligase (e.g. T4 DNA ligase ), the ends of the single strands are covalently linked to one another . In order to reduce the background of clones without inserts, the vector is often used to remove the 5 'phosphate group prior to ligation with phosphatase, e.g. B. calf intestine phosphatase (CIP) treated. This also allows efficient cloning in vectors that have only been treated with one restriction enzyme.

After the subsequent transformation, recombinant bacteria are selected by plating on agar plates with a suitable antibiotic . If the vectors allow, colonies that do not contain an insert are screened out by means of blue-white screening . This also does not guarantee that all other colonies contain the desired insert. Therefore, the DNA of individual colonies is characterized using restriction digestion or colony PCR . If the result is inconclusive or the insert was generated by DNA amplification , DNA sequencing is carried out . If one of the enzymes with which the vector is cut leaves a blunt end , the PCR fragment is cut with only one enzyme.

PCR cloning

In this variant, the DNA sequence to be inserted is amplified in a PCR using primers which each contain a sequence at the 5 'end that overlaps with the vector. The purified PCR product is in the course of ligation-during-amplification (PCR-related mutagenesis reaction) as a megaprimer used the plasmid in vitro to synthesize. Restriction enzymes and DNA ligase are not used here. After the starting plasmids have been broken down, the newly generated plasmids are transformed into bacteria for reproduction. The ligation takes place after the transformation in vivo . A variant of PCR cloning is circular polymerase extension cloning (CPEC).

TA cloning

The TA plasmids are also linearized plasmids, but they have a thymidine nucleotide as the last nucleotide as a 3 'overhang, which acts as a sticky end and makes a restriction of the DNA and the plasmid to be inserted unnecessary. The overhanging thymine nucleotide is previously attached by a deoxyribonucleotidyl transferase (English terminal deoxynucleotide transferase ) using dideoxy thymine nucleotides . The DNA sequence to be inserted must be generated with a thermostable DNA polymerase of type A (of bacterial origin), since type B polymerases do not create an overhang. After ligation, the plasmid is transformed into bacteria for multiplication. TA cloning avoids the use of restriction enzymes, but does not allow specific orientation of the insert in the vector, which means that gene expression can only take place in about 50% of the transgenic vectors. Therefore, TA vectors are often used in combination with a blue-white screening as pure cloning vectors .

TOPO cloning

TOPO plasmids are linearized by restriction digestion and then covalently coupled with a topoisomerase from the vaccinia virus (a pox virus ), which causes the ligation of a PCR product and therefore no longer requires a ligase. The covalent binding of the topoisomerase takes place automatically to a DNA sequence at the 3'-phosphate end of the sequence 5'- (C / T) CCTT-3 '. TOPO cloning vectors are available as TA vectors and as blunt-end vectors. The plasmid is then transformed into bacteria for reproduction. Since the orientation of the insert in the vector is random, these vectors are used as pure cloning vectors. If the first step is to generate an expression clone, unidirectional TOPO vectors are used. Unidirectional entry vectors are often used for the gateway system . TOPO and Gateway are registered trademarks of Thermo Fisher Scientific .

Isothermal assembly

By Isothermal Assembly be by PCR or artificial gene synthesis produced DNA fragments without the need to be treated with these restriction enzymes previously inserted into a linearized vector. The Gibson Assembly , which was developed by Daniel G. Gibson at the J. Craig Venter Institute , is often used . It allows the seamless cloning of one or more fragments into a vector in a single step . The prerequisite is that the molecules to be ligated have sequence overlaps of about 20 nucleotides . By incubation with dNTPs and a cocktail of three enzymes, an exonuclease ( T5 exonuclease ), which shortens the molecules from the 5 'end and thus allows the molecules to hybridize , a DNA polymerase ( Phusion DNA polymerase), the resulting Gaps are closed, and a thermostable DNA ligase ( Taq DNA ligase ), which causes a ring closure, creates plasmids that can be transformed directly. Gibson Assembly is a registered trademark of New England Biolabs .

LIC and SLIC

In another cloning variant ( ligation-independent cloning , LIC), the PCR product is provided with a LIC sequence. Using the 3 '→ 5' exonuclease activity of the T4 DNA polymerase , complementary overhangs are generated in the vector and in the PCR product in the presence of a dNTP . The ligation of the annealed product takes place after the transformation in vivo . A further development of the method is SLIC, the sequence and ligation-independent cloning that does not require a LIC sequence. The exonuclase activity is stopped in SLIC protocols by adding a dNTP. The gaps in the circular plasmids formed after mixing the DNA fragments are repaired after transformation into E. coli cells.

Gateway cloning

In a gateway cloning , sequences are added to the transgene that contain recognition sequences for the ligase attB , which catalyzes the incorporation of the transgene into an entry vector . At the same time, the suicide gene ccdB is removed from the entry vector , which means that only transgenic organisms with vectors grow. A subsequent exchange of gene segments by an excisionase results in a gene transfer from the entry vector into a target vector, which mostly serves to express the transgene and which has a different antibiotic resistance for later selection . As a second selection pressure, the entry vector receives the ccdB suicide gene from the target vector , which means that almost only organisms with the transgene-containing target vector grow.

Golden Gate cloning

The golden gate cloning , also known as the golden gate assembly , allows the simultaneous directional in vitro assembly of several DNA fragments with the help of type IIs restriction enzymes and T4 DNA ligase . The type IIs restriction enzymes used in this method, such as Bsa I, Bsm BI and Bbs I, cut outside of their recognition sequence and can therefore generate non-palindromic, four base-pair long overhangs that no longer have cleavage points after ligation. Since the restriction cleavage sites are not part of the resulting construct, restriction digestion and ligation can be carried out simultaneously. The method is u. a. used for the production of TALEN for genome editing .

Recombination

In the case of recombination processes such as recombineering and the RMCE cassette exchange process , restriction and ligation take place after the vector and insert have been transformed in vivo .

Artificial Gene Synthesis

The inserts can be synthesized de novo from primers by various methods of artificial gene synthesis . The synthesized double strands already contain the appropriate ends for ligation into the desired vector. This can e.g. B. protruding ends for ligation into a restriction enzyme cut vector or sequence overlaps for the Gibson assembly .

Transformation and Selection

Schematic representation of the transformation of a recombinant DNA into a bacterial cell and subsequent antibiotic selection.

This is followed by the transformation of competent bacterial cells (e.g. E. coli ) with the vector insert construct. The antibiotic - resistant gene on the vector allows for selection of bacterial cells that have taken up the plasmid into the cell. For this purpose, the transformation approach is on a agar - culture medium (eg. LB medium ) were plated, of the appropriate antibiotic (eg. Ampicillin or kanamycin containing). In this way, only those bacterial cells are selected that have taken up a plasmid. Bacterial colonies are formed by the multiplication of these individual bacterial cells . All untransformed bacteria that are not resistant to the antibiotic used die. If the vectors such. B. pUC19 allow this, colonies that contain no insert are screened out by means of blue-white screening . For this purpose, plates are used which, in addition to the antibiotic, also contain X-Gal and IPTG . The appearance of blue colonies indicates the presence of bacterial cells with unchanged vectors, while the unstained colonies may contain the desired insert. Therefore, the DNA of individual colonies is characterized either directly or after amplification in liquid culture, using restriction digestion or colony PCR . If the result is inconclusive or the insert was generated by DNA amplification , DNA sequencing is carried out . A liquid medium is inoculated with such a colony for further reproduction. A large amount of plasmid DNA can be isolated from such an approach ( plasmid preparation ). The DNA is then available for further cloning or transformation.

literature

Individual evidence

  1. ↑ J. Quan, J. Tian: Circular polymerase extension cloning of complex gene libraries and pathways. In: PloS one. Volume 4, number 7, 2009, p. E6441, ISSN  1932-6203 . doi: 10.1371 / journal.pone.0006441 . PMID 19649325 . PMC 2713398 (free full text).
  2. ↑ TA Holton, Graham, MW: A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors . In: Nucleic Acids Research . 19, No. 5, March 11, 1991, p. 1156. doi : 10.1093 / nar / 19.5.1156 . PMID 2020554 . PMC 333802 (free full text).
  3. ^ Y. Ichihara, Y. Kurosawa: Construction of new T vectors for direct cloning of PCR products. In: Gene (1993), vol. 130 (1), pp. 153-154. PMID 8344524 .
  4. ^ MY Zhou, CE Gomez-Sanchez: Universal TA cloning. In: Curr Issues Mol Biol. (2000), Volume 2 (1), pp. 1-7. PMID 11464915 .
  5. ↑ L. Geng, W. Xin, DW Huang, G. Feng: A universal cloning vector using vaccinia topoisomerase I. In: Mol Biotechnol. (2006), Volume 33 (1), pp. 23-28. PMID 16691003 .
  6. ↑ C. Cheng, S. Shuman: DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3 'mononucleotide overhang. In: Nucleic Acids Res. (2000), Vol. 28 (9), pp. 1893-1898. PMID 10756188 ; PMC 103307 (free full text).
  7. ↑ SG Morham, S. Shuman: Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I. In: J Biol Chem. (1992), Volume 267 (22), pp. 15984-15992. PMID 1322412 .
  8. ↑ J. Sekiguchi, S. Shuman: Proteolytic footprinting of vaccinia topoisomerase bound to DNA. In: J Biol Chem. (1995), Vol. 270 (19), pp. 11636-11645. PMID 7744804 .
  9. ↑ Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO .: Enzymatic assembly of DNA molecules up to several hundred kilobases . In: Nature Methods . 6, No. 5, 2009, pp. 343-345. doi : 10.1038 / nmeth.1318 . PMID 19363495 .
  10. ^ RM Benoit, C. Ostermeier, M. Geiser, JS Li, ​​H. Widmer, M. Auer: Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost. In: PloS one. Volume 11, number 4, 2016, p. E0153158, doi: 10.1371 / journal.pone.0153158 , PMID 27073895 , PMC 4830597 (free full text).
  11. ↑ RS Haun, IM Serventi, J. Moss: Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. In: BioTechniques (1992), Volume 13 (4), pp. 515-518. PMID 1362067 .
  12. ↑ MZ Li, SJ Elledge: Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. In: Nature methods. Volume 4, Number 3, March 2007, pp. 251-256, doi: 10.1038 / nmeth1010 , PMID 17293868 .
  13. ↑ D. Esposito, LA Garvey, CS Chakiath: Gateway cloning for protein expression. In: Methods in molecular biology. Volume 498, 2009, pp. 31-54, doi : 10.1007 / 978-1-59745-196-3_3 , PMID 18988017 .
  14. ↑ C. Engler, R. Kandzia, S. Marillonet: A One Pot, One Step, Precision Cloning Method with High Throughput Capability . In: PLOS One Volume 3 (11), 2008, e3647, PMID 18985154 .
  15. ^ E. Weber, C. Engler, R. Gruetzner, S. Werner, S. Marillonnet: A modular cloning system for standardized assembly of multigene constructs. In: PloS one. Volume 6, number 2, 2011, p. E16765, doi: 10.1371 / journal.pone.0016765 , PMID 21364738 , PMC 3041749 (free full text).
  16. ↑ C. Engler, R. Kandzia, S. Marillonnet: A one pot, one step, precision cloning method with high throughput capability. In: PloS one. Volume 3, number 11, 2008, p. E3647, doi: 10.1371 / journal.pone.0003647 , PMID 18985154 , PMC 2574415 (free full text).
  17. ^ NE Sanjana, L. Cong, Y. Zhou, MM Cunniff, G. Feng, F. Zhang: A Transcription Activator-Like Effector Toolbox for Genome Engineering . In: Nature Protocols Volume 7 (1), (2012), pp. 171-192. PMID 22222791 .
  18. ↑ Ulrich Helmich: Detection of cloned cells