Molecular cloning

Cloning ( or cloning, Eng. Molecular cloning ) is in the Molecular Biology of the umbrella term for methods of obtaining and identical reproduction of deoxyribonucleic acid ( DNA). Unlike cloning, whose aim is the production of genetically identical organisms, the cloning restricted to the manufacture of identical molecules of DNA.

Properties

In cloning a desired DNA fragment ( gene) integrated into a vector ( e.g. a plasmid or viral vector). The goal of cloning is to increase, a DNA fragment in order to investigate its properties or further use in further processes. After reproduction can be obtained by isolating total DNA is a multiple of the quantity of DNA used in the beginning, which is in contrast to low cost in vitro procedures such as PCR, precisely and in large numbers. Alternatively, the cells can express a gene product such as recombinant proteins (for example, a protein over-expression ). Such proteins play a role

• In biochemistry for further study of proteins and their functions
• To alter the properties of proteins in the context of a protein - engineering

• For therapeutic purposes (e.g., insulin)
• For use in food technology (eg rennet )
• For use in agriculture ( eg Flavr Savr tomato)

For the multiplication of cloning products different organisms are used as the host. Well-known examples are bacterial cells such as the bacterium Escherichia coli, single-celled algae or fungi. The host cells multiply it by cell division, whereby identical copies of the target DNA to be cloned are produced. The result is a population of cells a clone containing all of the desired DNA fragment. From this population, a suitable clone is isolated for further use.

Furthermore, the cloning can also be used to improve one or more genes to be transferred to other organisms, metabolic processes or to confer resistance ( animals and plants ), which leads to genetic manipulation. By a functional cloning similar DNA sequences are identified by a positional cloning adjacent DNA sequences are identified.

Method

When so-called cloning vectors ( " gene vectors " ) are used. These are used ( called transgene or English. Insert) as a vehicle for the transmission of a particular DNA sequence in a recipient cell. For the production of these vectors, there are various methods:

Restriction and ligation

The plasmid cloning a plasmid (eg pBR322 ) cut mixed with the help of special restriction enzymes so that overhanging ends ( engl. sticky ends, sticky ends ) arise. The target DNA was amplified in a polymerase chain reaction ( PCR) from genomic DNA and then to be incorporated as an insert into the vector is cut with the same enzymes, so that complementary ends on vector and the target DNA produced. The mutually compatible overhanging ends of vector and target DNA can be found and hybridize with each other. In the subsequent ligation, the (eg T4 DNA ligase ), catalyzed by a DNA ligase to the ends of the single strands are linked covalently to each other.

In the ligation take by far not all plasmids the DNA fragment to be multiplied on. Therefore, a further selection step is needed. Such unmodified plasmids may be due to early closure of the plasmid or more simply, the non-inclusion of a DNA fragment in the polylinker region. Since only the plasmids are of interest for the remainder containing the insert to be multiplied, these useless vectors must be discarded after transformation by selection.

PCR cloning

In this variant, the inserted DNA sequence is amplified in a PCR with primers that contain an overlapping with the vector sequence at the 5 'end. The purified PCR product ( a PCR - related mutagenesis reaction ) is used as part of a ligation -during -amplification as a megaprimer to synthesize the plasmid in vitro. Restriction enzymes and DNA ligase are not used here. After a reduction of Ausgangsplasmide the newly generated plasmids are transformed for propagation in bacteria. The ligation is carried out after the transformation in vivo.

TOPO cloning

TOPO - plasmids are linearized by restriction digestion and then covalently coupled to a topoisomerase from the vaccinia virus ( smallpox virus) which causes the ligation of the PCR product and thus requires no ligase. The covalent binding of the topoisomerase is made to a DNA sequence at the 3'-end (5'- (C / T) CCTT -3 '). Subsequently, the plasmid is transformed for replication in bacteria. TOPO cloning avoids use of restriction enzymes, but does not allow selective orientation of the insert in the vector, which gene expression can take place in only about 50 % of the transgenic vector.

TA cloning

TA plasmids also linearized plasmids, which however have a thymidine nucleotide as a 3'-overhang as the last nucleotide, which acts as a sticky end and eliminates a restriction of the inserted DNA and the plasmid. The overhanging thymine nucleotide is added by a terminal deoxynucleotidyl previously ( engl. terminal deoxynucleotides transferase ) using dideoxy Thyminnukleotiden. The inserted DNA sequence must in this case be produced with a thermostable DNA polymerase, A-type ( of bacterial origin ). After ligation, the plasmid is transformed for propagation in bacteria. TA cloning avoids use of restriction enzymes, but does not allow selective orientation of the insert in the vector, which gene expression can take place in only about 50 % of the transgenic vector.

LIC cloning

In a further variant (English ligation -independent cloning, LIC), the PCR product is provided with a LIC sequence. The ligation is carried out after the transformation in vivo.

Gateway cloning

A Gateway cloning sequences are appended to the transgene containing recognition sequences for the attB ligase, which catalyzes the incorporation of the transgene into an entry vector. Here is simultaneously removed the ccdB suicide gene in the entry vector, which grow only transgenic organisms. By a subsequent exchange of gene segments with an excisionase gene transfer vector is carried by the entry into a target vector which is mostly of the expression of the transgene, and for later has a different antibiotic resistance selection. As a second selection pressure obtained with this exchange of entry vector from the target vector, the ccdB suicide gene, which grow almost only organisms with the transgene -containing destination vector.

Recombination

In the recombination, such as the recombineering and RMCE cassette exchange procedure, the restriction and ligation after joint transformation of vector and insert takes place in vivo.

Artificial gene synthesis

Through various methods of the artificial gene synthesis, the inserts may be synthesized de novo from primers and inserted by PCR-based cloning into the vector.

Transformation and Selection

Subsequently, the transformation of competent bacterial cells (such as E. coli) with the vector-insert construct follows. The selection of cells that have actually taken up the chimera, is now carried out in two steps. First, the bacterial cells that have taken up no plasmid, killed. Has a bacterial cell, the plasmid was added to the cell interior, it can with the aid of an existing on the plasmid antibiotic resistance gene on an agar medium ( for example, LB medium or YT medium ) waxes of the appropriate antibiotic (eg contains. ampicillin or kanamycin ). Thus, only those bacteria cells are selected that have taken up a plasmid. By multiplication of these individual bacterial cell bacterial colonies arise. All untransformed and thus not die against the used antibiotic resistant bacteria.

In the second step, the selection takes place by blue - white selection and / or by colony PCR, being located in the following specifically related to the vector pUC18 and its derivatives are often used. His polylinker region located on the lacZ gene encoding the enzyme β -galactosidase. In the unmodified plasmid, this enzyme is expressed and can be split hydrolytically able to β -glucosidic bonds of the galactose. However, this vector is transformed to the inserted DNA fragment interrupts said gene, whereby no more active β -galactosidase can be expressed. In the case of said vector, this phenomenon is used by the use of the colorless X-gal glycoside. Its aqueous solution is applied to the - coated bacterial cells - remaining after the first selection. The molecules of the X - Gal comprised of β -D -galactose, which is bound to an indigo derivative. Now take the bacterial cells which contain several untransformed plasmids of this material, the bond between the β -galactose and the indigo derivative of the expremierte enzyme β -galactosidase is cleaved, so that the unbound blue coloring, indigo derivative is formed on or off. The formation of blue colonies indicates the presence of bacteria cells with vectors unchanged while the unstained colonies potentially containing the gene, since the enzyme is inactive in a vector with an insert.

To further increase the inoculation with such a colony to a liquid medium. From such an approach, a large amount of plasmid DNA was isolated ( plasmid ) are. The DNA is then available for further cloning or transformations available.